Human Diaphragms Research Articles

A bilinear structural constitutive model for strain rate-dependent behaviour of human diaphragm tissue

Diaphragmatic rupture is the tear of the diaphragm muscle result from blunt or penetrating trauma and occurs in about 5% of cases of severe blunt trauma. Both finite element (FE) modelling and experimental testing have enhanced our understanding of the injury mechanisms associated with diaphragmatic rupture. A constitutive model for human diaphragm tissue is developed and implemented via user subroutine (UMAT) in LS Dyna that accounts for the strain rate-dependent effects and bilinear behaviour observed experimentally. To better understand the material properties of the human diaphragm, 16 dynamic tensile tests were conducted at three different strain rates of 65/s, 130/s and 190/s from six whole human diaphragms. The engineering stress–strain relationship obtained from these tests showed a bilinear behaviour and strain rate dependency. A strain rate-dependent bilinear stress–strain model was developed, and its parameters were optimised using a genetic algorithm-based inverse characterization method. The results demonstrate a good correlation between experiments and the model, with an average difference of 2 ± 2.8% (mean ± SD) between the optimised FE and experimental load–time curves. The material parameters found in this study can be used in dynamic simulations using FE models of diaphragm tissues.

Mesenchymal expression of the FRAS1/FREM2 gene unit is decreased in the developing fetal diaphragm of nitrofen-induced congenital diaphragmatic hernia.

Developmental mutations that inhibit normal formation of extracellular matrix (ECM) in fetal diaphragms have been identified in congenital diaphragmatic hernia (CDH). FRAS1 and FRAS1-related extracellular matrix 2 (FREM2), which encode important ECM proteins, are secreted by mesenchymal cells during diaphragmatic development. The FRAS1/FREM2 gene unit has been shown to form a ternary complex with FREM1, which plays a crucial role during formation of human and rodent diaphragms. Furthermore, it has been demonstrated that the diaphragmatic expression of FREM1 is decreased in the nitrofen-induced CDH model. We hypothesized that FRAS1 and FREM2 expression is decreased in the developing diaphragms of fetal rats with nitrofen-induced CDH. Pregnant rats were exposed to either nitrofen or vehicle on gestational day 9 (D9), and fetuses were harvested on D13, D15 and D18. Microdissected diaphragms were divided into nitrofen-exposed/CDH and control samples (n=12 per time-point and experimental group, respectively). Diaphragmatic gene expression levels of FRAS1 and FREM2 were analyzed by qRT-PCR. Immunofluorescence double staining for FRAS1 and FREM2 was combined with the mesenchymal marker GATA4 in order to evaluate protein expression and localization in pleuroperitoneal folds (PPFs) and fetal diaphragmatic tissue. Relative mRNA expression of FRAS1 and FREM2 were significantly reduced in PPFs of nitrofen-exposed fetuses on D13 (1.76±0.86 vs. 3.09±1.15; p<0.05 and 0.47±0.26 vs. 0.82±0.36; p<0.05), developing diaphragms of nitrofen-exposed fetuses on D15 (1.45±0.80 vs. 2.63±0.84; p<0.05 and 0.41±0.16 vs. 1.02±0.49; p<0.05) and fully muscularized diaphragms of CDH fetuses on D18 (1.35±0.75 vs. 2.32±0.92; p<0.05 and 0.37±0.24 vs. 0.70±0.32; p<0.05) compared to controls. Confocal laser scanning microscopy revealed markedly diminished FRAS1 and FREM2 immunofluorescence in diaphragmatic mesenchyme, which was associated with reduced proliferation of mesenchymal cells in nitrofen-exposed PPFs and fetal CDH diaphragms on D13, D15 and D18 compared to controls. Decreased mesenchymal expression of FRAS1 and FREM2 in the nitrofen-induced CDH model may cause failure of the FRAS1/FREM2 gene unit to activate FREM1 signaling, disturbing the formation of diaphragmatic ECM and thus contributing to the development of diaphragmatic defects in CDH.

The clinical anatomy of the musculotendinous part of the diaphragm.

The thoracoabdominal diaphragm is a composite musculotendinous structure, separating the thoracic and abdominal cavities. Reemphasis of the already well-delineated variations of the muscular and tendinous portions, and blood and nerve supply of the diaphragm is becoming apparent. Scientific reports concerning reconstruction of the pericardium, activation of the muscle and the phrenic nerves by use of laparoscopically placed intramuscular electrodes, and repair of congenital and traumatic hernias reemphasize the importance of the muscular to tendinous relationships. The objective of this study, therefore, was to measure the ratio of the surface area of the tendinous central region to the muscular region of the diaphragm and provide a clear description across various specimens. We classified diaphragmatic measurements from 104 adult human diaphragms into six classes (I-VI) based on the ratio of surface area between its tendinous and muscular components. The majority of specimens, 56.7%, was attributed to class II and indicated a tendon-to-muscle ratio of between 10 and 15%; however, a small number of specimens indicated a very large tendon area at the expense of muscle bulk. Future research should be geared toward assessing the relationship between surface area of the musculature and its motor points with focus on interventions for herniation repair and recovery. Our results have shown that surgical interventions should be tailored to the individual, as diaphragm size may not necessarily predict tendon-to-muscle ratio.

The neonatal sarcoplasmic reticulum Ca2+-ATPase gives a clue to development and pathology in human muscles.

The sarcoplasmic/endoplasmic reticulum calcium ATPase 1 (SERCA1) has two muscle specific splice isoforms; SERCA1a in fast-type adult and SERCA1b in neonatal and regenerating skeletal muscles. At the protein level the only difference between these two isoforms is that SERCA1a has C-terminal glycine while SERCA1b has an octapeptide tail instead. This makes the generation of a SERCA1a specific antibody not feasible. The switch between the two isoforms is a hallmark of differentiation so we describe here a method based on the signal ratios of the SERCA1b specific and pan SERCA1 antibodies to estimate the SERCA1b/SERCA1a dominance on immunoblot of human muscles. Using this method we showed that unlike in mouse and rat, SERCA1b was only expressed in pre-matured infant leg and arm muscles; it was replaced by SERCA1a in more matured neonatal muscles and was completely absent in human foetal and neonatal diaphragms. Interestingly, only SERCA1a and no SERCA1b were detected in muscles of 7-12years old boys with Duchenne, a degenerative-regenerative muscular dystrophy. However, in adult patients with myotonic dystrophy type 2 (DM2), the SERCA1b dominated over SERCA1a. Thus the human SERCA1b has a different expression pattern from that of rodents and it is associated with DM2.

Mitochondrial Dysfunction and Lipid Accumulation in the Human Diaphragm during Mechanical Ventilation

Mechanical ventilation (MV) is associated with adverse effects on the diaphragm, but the cellular basis for this phenomenon, referred to as ventilator-induced diaphragmatic dysfunction (VIDD), is poorly understood. To determine whether mitochondrial function and cellular energy status are disrupted in human diaphragms after MV, and the role of mitochondria-derived oxidative stress in the development of VIDD. Diaphragm and biceps specimens obtained from brain-dead organ donors who underwent MV (15-176 h) and age-matched control subjects were compared regarding mitochondrial enzymatic function, mitochondrial DNA integrity, lipid content, and metabolic gene and protein expression. In addition, diaphragmatic force and oxidative stress after exposure to MV for 6 hours were evaluated in mice under different conditions. In human MV diaphragms, mitochondrial biogenesis and content were down-regulated, with a more specific defect of respiratory chain cytochrome-c oxidase. Laser capture microdissection of cytochrome-c oxidase-deficient fibers revealed mitochondrial DNA deletions, consistent with damage from oxidative stress. Diaphragmatic lipid accumulation and responses of master cellular metabolic sensors (AMP-activated protein kinase and sirtuins) were consistent with energy substrate excess as a possible stimulus for these changes. In mice, induction of hyperlipidemia worsened diaphragmatic oxidative stress during MV, whereas transgenic overexpression of a mitochondria-localized antioxidant (peroxiredoxin-3) was protective against VIDD. Our data suggest that mitochondrial dysfunction lies at the nexus between oxidative stress and the impaired diaphragmatic contractility that develops during MV. Energy substrate oversupply relative to demand, resulting from diaphragmatic inactivity during MV, could play an important role in this process.

Intramuscular distribution of the phrenic nerve in human diaphragm as shown by Sihler staining

Intramuscular innervation of the human diaphragm has not been well described. The goal of this study was to elucidate the detailed intramuscular distribution of the phrenic nerve in the human diaphragm. Fifteen human diaphragms were visualized using modified Sihler staining, and the detailed intramuscular phrenic nerve distribution was photographed and recorded. Three types of primary phrenic nerve branches were noted. Each type of primary branch innervated a confined muscular subvolume of the diaphragm, and the intramuscular branches in each subvolume anastomosed largely with one another and formed a characteristic "net" of nerve branches. A few small nerve filaments were seen entering the peripheral diaphragm. The directions and locations of nerve branches innervating the hiatal diaphragm were not symmetrical between sides. These findings may offer useful information for anatomists, physiologists, and clinicians.

Respiratory muscle fiber remodeling in chronic hyperinflation: dysfunction or adaptation?

The diaphragm and other respiratory muscles undergo extensive remodeling in both animal models of emphysema and in human chronic obstructive pulmonary disease, but the nature of the remodeling is different in many respects. One common feature is a shift toward improved endurance characteristics and increased oxidative capacity. Furthermore, both animals and humans respond to chronic hyperinflation by diaphragm shortening. Although in rodent models this clearly arises by deletion of sarcomeres in series, the mechanism has not been proven conclusively in human chronic obstructive pulmonary disease. Unique characteristics of the adaptation in human diaphragms include shifts to more predominant slow, type I fibers, expressing slower myosin heavy chain isoforms, and type I and type II fiber atrophy. Although some laboratories report reductions in specific force, this may be accounted for by decreases in myosin heavy chain content as the muscles become more oxidative and more efficient. More recent findings have reported reductions in Ca(2+) sensitivity and reduced myofibrillar elastic recoil. In contrast, in rodent models of disease, there is no consistent evidence for loss of specific force, no consistent shift in fiber populations, and atrophy is predominantly seen only in fast, type IIX fibers. This review challenges the hypothesis that the adaptations in human diaphragm represent a form of dysfunction, secondary to systemic disease, and suggest that most findings can as well be attributed to adaptive processes of a complex muscle responding to unique alterations in its working environment.

Developmental myosin heavy chains in the adult human diaphragm: coexpression patterns and effect of COPD.

In preliminary experiments we noted developmental (i.e., embryonic and neonatal) myosin heavy chains (MHCs) in the diaphragms of patients with severe chronic obstructive pulmonary disease (COPD). We hypothesized that this finding represented new fiber formation secondary to injury associated with the mechanical stress of COPD or previously undescribed MHCs in the human diaphragm. To distinguish between these possibilities, we analyzed diaphragmatic biopsies obtained from 9 patients with severe COPD (forced expiratory volume in 1 s = 21 +/- 2% predicted, residual volume = 283 +/- 22% predicted) and 10 age-matched controls. First, using immunocytochemistry with specific monoclonal antibodies, we noted that control diaphragms had greater proportions of fibers expressing embryonic (50 +/- 2 vs. 28 +/- 3%, P < 0.0001) and neonatal (52 +/- 2 vs. 32 +/- 3%, P < 0.001) MHCs than COPD diaphragms. Second, SDS-PAGE demonstrated that these developmental MHCs represented only a very small fraction of the diaphragmatic MHC content. Third, the RT-PCR demonstrated mRNA coding for embryonic and neonatal MHCs in COPD and control diaphragms. Last, COPD and control diaphragms exhibited normal histology on light microscopy. We conclude that the presence of developmental MHC isoforms does not indicate new fiber formation in diaphragms of patients with severe COPD. Although these results represent the first systematic description of embryonic and neonatal MHCs in normal adult human diaphragms, their function remains to be elucidated.

Expression and regulation of protein inhibitor of neuronal nitric oxide synthase in ventilatory muscles.

In skeletal muscle fibers, nitric oxide (NO) is synthesized by neuronal NO synthase (nNOS) and regulates excitation-contraction coupling, glucose uptake, and mitochondrial respiration. Recently, a novel 89-amino acid protein, designated protein inhibitor of nNOS (PIN), has been shown to interact with and specifically inhibit nNOS activity. In this study, we investigated the distribution, localization, and regulation of PIN expression in ventilatory and limb muscles of various species. Amplified PIN cDNA from the rat diaphragm revealed an open reading frame identical to that of human PIN. Among muscles of adult rats, PIN mRNA was strongly expressed in muscles rich in type I fibers, whereas much weaker expression was evident in muscles rich in type II fibers. By comparison, PIN protein expression was not related to fiber-type distribution. Similarly, PIN protein was equally expressed among rat, mouse, and human diaphragms. Both PIN mRNA and PIN protein were expressed at much higher levels in the embryonic rat diaphragm than in adult muscle. Immunohistochemistry revealed that PIN protein was localized in close proximity to the sarcolemma and nuclei. PIN protein was also abundant in muscle spindles and axons of nerves supplying skeletal muscle fibers. We conclude that PIN is expressed in various skeletal muscle fibers and that its expression is regulated during muscle development. The localization of PIN in muscle regions containing abundant nNOS protein suggests that it plays a role in the regulation of NO synthesis in skeletal muscle fibers.

Ulceration, fibrosis and diaphragm‐like lesions in the caecum of rats treated with indomethacin

Patients on nonsteroidal anti-inflammatory drugs can develop curious intestinal fibrotic diaphragms. Groups of rats received indomethacin mixed into a powdered diet at 3 mg.kg/day for 6 and 12 weeks and 6 mg.kg/day for up to 6 weeks. In an attempt to reproduce a human dosing regimen, another group of rats, for a total of 30 weeks, received consecutive periods of indomethacin at 3 mg.kg/day for 12 weeks, 4.5 mg.kg/day for 1 week, 6 mg.kg/day for 1 week, control diet for 6 weeks, 4.5 mg.kg/day for 2 weeks and finally, a control diet for a healing period of 8 weeks. Control rats received powdered diet alone. At termination, the small and large intestines were examined macroscopically and histologically. Indomethacin caused microcytic anaemia, hypoalbuminaemia, small intestinal ulceration, caecal ulceration and inconspicuous raised mucosal lesions in the caecum that histologically showed submucosal fibrosis with disruption and thickening of the apical muscularis mucosae. No control rats showed any abnormality. These fibrotic lesions of the rat caecum resemble human diaphragms and may arise from healed caecal ulcers.

Choline ester hydrolases in diaphragm muscle

By the use of centrifugal fractionation, specific substrates and selective inhibitors evidence was obtained that the diaphragm muscle of guinea pig, man and rat contain both soluble and particulate acetylcholinesterase (EC 3.1.1.7) and a soluble cholinesterase (EC 3.1.1.8). This is believed to be the first report of naturally-occurring soluble mammalian acetylcholinesterases. Human and rat diaphragms also contain a physostigmine-resistant enzyme which hydrolyses butyrylcholine, and is probably a B-esterase. It was noted incidentally that a modified Koelle histochemical method, using thiocholine esters, has a specific requirement for iodide ion which has not previously been reported.

Trichiniasis in Man and Animals in Cincinnati, Ohio

Human diaphragms, swine diaphragms, pork products, and wild animals obtained in Cincinnati, Ohio, were examined for the presence of Trichinella spiralis. Larvae were found by means of standard methods of direct microscopic observation and artificial digestion. Of 332 human diaphragms obtained at necropsy 16 proved positive for the presence of T. spiralis, and five of 700 grainand garbage-fed hogs were infected. No evidence of T. spiralis infection was found in either the pork products or wild animals examined. Many studies on the incidence of Trichinella spiralis in man and swine have been conducted in the United States, but none have been done in the Cincinnati area. Most (1965) reviewed studies on human trichiniasis and reported an apparently significant reduction in incidence, mortality, and density of infection in man since 1944, and stated that the adult infection rate in the U. S. is probably 4% or less. Zimmermann and Brandly (1965) reviewed studies on the incidence of T. spiralis in swine, and noted a decline in incidence during the period of their study. Bachman and Rodriguez-Molina (1933) stated that T. spiralis is among the most euryxenous of nematodes. A great deal of controversy has arisen with respect to the importance of reservoir hosts in the epidemiology of the parasite. Among others, Most (1965), Gould (1943), and Wright (1940) discount the possibility of reservoir hosts as a source of infection for swine. On the other hand, Schwartz (1960) considered the role of reservoir hosts as uncertain, and several workers, including Spindler (1953), Olson and Robinson (1958), and Zimmermann et al. (1959), have demonstrated transmission of T. spiralis through the feces of infected animals to clean animals. In view of this experimental evidence of transmission through fecal contamination, and the availability of infected wild animal carcasses to swine, reservoir hosts remain an important possible factor in the epidemiology of T. spiralis. In an attempt to demonstrate a relationship between infected swine and wild animals, Martin et al. (1968) studied two Illinois swine farms. The swine from one farm were fed garbage, and had a history of T. spiralis infection, while the swine Received for publication 16 October 1968. of the other farm were fed grain. Since only two wild animals from each farm were found infected with T. spiralis, no conclusions were drawn regarding the effects of wild animals on the epidemiology of the parasite. MATERIALS AND METHODS Fresh human diaphragm samples were obtained from four Cincinnati hospitals and the Hamilton County Coroner's Office from October 1966 to August 1967. About one-half a diaphragm was collected from randomly selected cadavers at each hospital and data on age, sex, and race were obtained from hospital records whenever possible. All samples were examined by microscopic observation of pressed muscle, and by artificial digestion in 1% pepsin, 1% HC1 in 0.85% NaCl. The sediment from each digested sample was examined under a dissecting microscope. Swine diaphragm samples were obtained from a Cincinnati meat-packing company from January 1966 to September 1966. The swine used by this concern are a mixture of garbage-fed and grainfed animals, but it was not possible to divide the samples on the basis of feed. Swine diaphragm tissue was examined by artificial digestion only. Pork sausage was obtained from the company that provided the diaphragm samples, and from another Cincinnati meat-packing company. The pork products were examined by artificial digestion. A limited number of local animals were examined for T. spiralis larvae. More than 40 rats were trapped at the Cincinnati Zoo, and some commercially valuable fur-bearing animals were obtained from a Cincinnati furrier. The latter animals were taken from nearby areas, but exact collection data were not available. All animals collected were examined by artificial digestion of the diaphragm, tongue, and leg muscles.

EXAMINATION OF HUMAN DIAPHRAGMS FOR TRICHINOSIS IN HIROSHIMA AND NAGASAKI

In view of the finding of Trichinella spira spiralis in three species of mammals born in Hokkaido, it is thought that the parasite may also infect other species of animals in Japan, including man. Although examination of human diaphragms in a small series of 149 autopsies in Hiroshima and 37 autopsies in Nagasaki (by digestion, compression, and microscopic sections) failed to reveal any instance of trichinous infection, it is that the existence or absence of human trichinosis in Japan can be determined only by examination covering a larger series of autopsies, and performed in various parts of the country. ACTA PATH. JAP.18: 245–247, 1968.

Xenodiagnostic technic as an aid in diagnosis of trichinosis.

SummaryA study was made of 100 human diaphragms in Mexico City to determine the incidence and degree of infection with T. spiralis.Direct microscopic examination of samples weighing 50 gm. or more revealed a very high incidence (15 per cent) of low grade infections. Twelve of the 15 positive cases showed less than 1 cyst per gm. of tissue, while 9 showed less than 1 cyst per 10 gm. of tissue.Xenodiagnosis, using albino rats, was used to detect viability in the positive cases. This method is proposed as a means of detecting all cases having viable larvae in future survey work.

Incidence of Trichinella infections in San Francisco, 1950.

About 20 years ago interest in human trichinosis in the United States was stimulated by Queen's work, 1 in which he showed the incidence of infections, as measured by recovery of larvae from human diaphragms by peptic digestion, to be far higher than would be indicated by the reported diagnoses of clinical trichinosis. His study was followed by those of a succession of investigators who used similar techniques. Extensive studies of trichinous infections in men and in hogs were made, notably by Hall and Collins, and others working with Willard Wright at the National Institute of Health. 2 It was established that infections by Trichinella spiralis were quite common. Larvae were found in about 16 per cent of the human diaphragms collected in various parts of the United States. Wright, Jacobs and Walton 3 concluded that there was no correlation between Trichinella infection and sex, civil or military status, past