Sensory receptors in the visceral pleura: neurochemical coding and live staining in whole mounts

Today, diagnosis and treatment of chest pain related to pathologic changes in the visceral pleura are often difficult. Data in the literature on the sensory innervation of the visceral pleura are sparse. The present study aimed at identifying sensory end-organs in the visceral pleura, and at obtaining more information about neurochemical coding. The immunocytochemcial data are mainly based on whole mounts of the visceral pleura of control and vagally denervated rats. It was shown that innervation of the rat visceral pleura is characterized by nerve bundles that enter in the hilus region and gradually split into slender bundles with a few nerve fibers. Separate nerve fibers regularly give rise to characteristic laminar terminals. Because of their unique association with the elastic fibers of the visceral pleura, we decided to refer to them as "visceral pleura receptors" (VPRs). Cryostat sections of rat lungs confirmed a predominant location on mediastinal and interlobar lung surfaces. VPRs can specifically be visualized by protein gene product 9.5 immunostaining, and were shown to express vesicular glutamate transporters, calbindin D28K, Na+/K+-ATPase, and P2X3 ATP-receptors. The sensory nerve fibers giving rise to VPRs appeared to be myelinated and to have a spinal origin. Because several of the investigated proteins have been reported as markers for sensory terminals in other organs, the present study revealed that VPRs display the neurochemical characteristics of mechanosensory and/or nociceptive terminals. The development of a live staining method, using AM1-43, showed that VPRs can be visualized in living tissue, offering an interesting model for future physiologic studies.     

Elastic Behavior of Porcine Coronary Artery Tissue Under Uniaxial and Equibiaxial Tension

The aim of this study was to characterize the nonlinear anisotropic elastic behavior of healthy porcine coronary arteries under uniaxial and equibiaxial tension. Porcine coronary tissue was chosen for its availability and similarity to human arterial tissue. A biaxial test device previously used to test human femoral arterial tissue samples (Prendergast, P. J., C. Lally, S. Daly, A. J. Reid, T. C. Lee, D. Quinn, and F. Dolan. ASME J. Biomech. Eng., Vol. 125, pp. 692-699, 2003) was further developed to test porcine coronary tissue specimens. The device applies an equal force to the four sides of a square specimen and therefore creates a biaxial stretch that demonstrates the anisotropy of arterial tissue. The nonlinear elastic behavior was marked in both uniaxial and biaxial tests. The tissue demonstrated higher stiffness in the circumferential direction in four out of eight cases subjected to biaxial tension. Even though anisotropy is demonstrated it is proposed that an isotropic hyperelastic model may adequately represent the properties of an artery, provided that an axial stretch is applied to the vessel to simulate the in vivo longitudinal tethering on the vessel. Isotropic hyperelastic models based on the Mooney-Rivlin constitutive equation were derived from the test data by averaging the longitudinal and circumferential equibiaxial data. Three different hyperelastic models were established to represent the test specimens that exhibited a high stiffness, an average stiffness, and a low stiffness response; these three models allow the analyst to account for the variability in the arterial tissue mechanical properties. These models, which take account of the nonlinear elastic behavior of coronary tissue, may be implemented in finite element models and used to carry out preclinical tests of intravascular devices. The errors associated with the hyperelastic models when fitting to both the uniaxial and equibiaxial data for the low stiffness, average stiffness, and high stiffness models were found to be 0.836, 5.206, and 2.980, respectively.     

Biaxial Testing of Human Annulus Fibrosus and Its Implications for a Constitutive Formulation

Internal pressure in the healthy human annulus fibrosus leads to multiaxial stress in vivo, yet uniaxial tests have been used exclusively to characterize its in vitro mechanical response and to determine its elastic strain energy function (W). We expected that biaxial tension tests would provide unique and necessary data for characterizing the annular material response, and thereby, for determining W. We performed uniaxial and biaxial tests on specimens of annulus, then developed an objective methodology for defining an appropriate form for W that considers data from multiple experiments simultaneously and allows the data to dictate more directly the form and the number of parameters needed. We found that the stresses attained in the biaxial tests were higher, while the strains were considerably lower, than those observed in the uniaxial tests. A comparison of strain energy functions determined from the different data sets demonstrated that constitutive models derived from uniaxial data could not predict annulus behavior in biaxial tension and vice versa. Since the annulus is in a state of multaxial stress in vivo, we conclude that uniaxial tests alone are insufficient to prescribe a physiologically relevant W for this tissue.     

Effect of adrenaline on transmesothelial resistance of isolated sheep pleura

The effect of adrenaline on the transmesothelial resistance (RTM) of sheep's visceral and parietal pleura was studied using the Ussing chamber technique. Basal transmesothelial resistance of visceral pleura was found to be 20.71 +/- 0.31 Omega cm2, whereas that of parietal pleura was found to be 19.53 +/- 0.34 Omega cm2. Immediately after the addition of adrenaline (10(-7) M) both apically and basolaterally on the visceral and parietal pleura, these values were significantly increased (P < 0.05). Addition of the nonselective beta-receptor blocker, propranolol (10(-5) M), suppressed this effect in both visceral and parietal pleura, while addition of the nonselective alpha-receptor blocker, phentolamine (10(-5) M), partly suppressed the above-mentioned increase in the parietal pleura. In conclusion, our results show that adrenaline has a rapid effect on both pleurae. This rapid effect is mediated by the stimulation of beta-adrenergic receptors in the case of visceral pleura, while in the case of parietal pleura this effect seems to be due to a stimulation of alpha- and beta-adrenergic receptors. On the visceral pleura the effect of adrenaline vanishes after some minutes and on the parietal this effect is more permanent than the visceral's one, suggesting differences in the distribution of the adrenergic receptors between the visceral and parietal pleura.     

Experimental and numerical analyses of indentation in finite-sized isotropic and anisotropic rubber-like materials

Indentation tests perpendicular to the major plane of a material have been proposed as a means to index some of its in-plane mechanical properties. We showed the feasibility of such tests in myocardial tissue and established its theoretical basis with a formulation of small indentation superimposed on a finitely stretched half-space of isotropic materials. The purpose of this study is to better understand the mechanics of indentation with respect to the relative effects of indenter size, indentation depth, and specimen size, as well as the effects of material properties. Accordingly, we performed indentation tests on slabs of silicone rubber fabricated with both isotropic, as well as transversely isotropic, material symmetry. We performed indentation tests in different thickness specimens with varying sizes of indenters, amounts of indentation, and amounts of in-plane stretch. We used finite-element method simulations to supplement the experimental data. The combined experimental and modeling data provide the following useful guidelines for future indentation tests in finite-size specimens: (i) to avoid artifacts from boundary effects, the in-plane specimen dimensions should be at least 15 times the indenter size; (ii) to avoid nonlinearities associated with finite-thickness effects, the thickness-to-radius ratio should be >10 and thickness to indentation depth ratio should be >5; and (iii) we also showed that combined indentation and inplane stretch could distinguish the stiffer direction of a, transversely isotropic material.     

Design of a novel MEMS platform for the biaxial stimulation of living cells

Micromechanical systems are increasingly being used as tools in biological applications, since their characteristic dimensions permit to operate at the same length scale of the structures under investigation. Here, we present a methodology for the design, fabrication and operation of a tool for the assessment of mechanical properties of single cells. In particular, we describe a microsystems platform to study bio-mechanical response of single living cells to in-plane biaxial stretching. The proposed device employs a new linkage design in order to obtain the displacement of the quadrants of a sliced circular plate in mutually-orthogonal directions using just one linear actuator. With this linkage geometry, the whole device has only one degree of freedom. This results in a very predictable and reliable mechanical behaviour, thereby allowing use a simple and easily available control electronics. Results of this study have relevance for the design of a powerful yet simple BioMEMS platform for the characterization of living cells as in-plane bi-axial loading simulated the conditions experienced by cells in vivo more realistically than a uniaxial stretching.     

小白鼠胸膜间皮的扫描电镜观察

目的　观察小白鼠脏、壁胸膜间皮细胞在扫描电镜下的结构以及两者在结构上的异同。方法　用扫描电镜对比观察 5只小白鼠的脏胸膜及各部壁胸膜的结构。结果　脏、壁胸膜间皮细胞表面有大量粗、细微绒毛 ,较多的火山口样凹陷及一些细胞间孔 ;脏胸膜间皮表面粗微绒毛密集且分支多 ,分支内有大量串珠样颗粒 ,多见具有芽状或颗粒状微绒毛的幼稚间皮细胞 ;壁胸膜间皮表面粗微绒毛稀疏 ,细微绒毛密集 ,多见微绒毛退化消失且有即将与间皮分离的衰老细胞、火山口样凹陷及细胞间孔。结论　脏胸膜间皮可能具有较强分泌功能 ,而壁胸膜间皮吸收功能较强     

Tight junction proteins contribute to barrier properties in human pleura

The permeability of pleural mesothelium helps to control the volume and composition of the liquid lubricating pleural surfaces. Information on pleural barrier function in health and disease, however, is scarce. Tissue specimens of human pleura were mounted in Ussing chambers for measurement of transmesothelial resistance. Expression of tight junction (TJ) proteins was studied by Western blots and immune fluorescence confocal microscopy. Both visceral and parietal pleura showed barrier properties represented by transmesothelial resistance. Occludin, claudin-1, -3, -5, and -7, were detected in visceral pleura. In parietal pleura, the same TJ proteins were detected, except claudin-7. In tissues from patients with pleural inflammation these tightening claudins were decreased and in visceral pleura claudin-2, a paracellular channel former, became apparent. We report that barrier function in human pleura coincides with expression of claudins known to be key determinants of epithelial barrier properties. In inflamed tissue, claudin expression indicates a reduced barrier function.     

Rare cause of pleural nodularity: Splenosis

Splenosis is a rare condition described as ectopic splenic tissue implantation generally after a splenic rupture. A 35-year-old male patient who had a history of splenectomy operation due to gunshot wound seven years ago was referred to our hospital with complaints, including exhaustion, sweating and shortness of breath. Thoracic computed tomography of the patient showed nodular pleural thickenings at the level of the left lower lobe, which proved to be unrelated with the presenting symptoms. The patient underwent a minithoracotomy for diagnosis and treatment. During the intraoperative observation, dark-colored, soft, multiple nodular lesions with a biggest size of 2 cm inside the visceral pleura over an area of 5 x 10 cm in dimension were observed. Also, a few tiny nodules in the lung parenchyma approximately 1-cm deep to the pleural nodules were palpated. The lesions were excised. The histopathological examination of the specimen showed splenic tissue in the lung parenchyma and pleura, so the lesions were accepted as splenosis. Pleuropulmonary splenosis, which develops generally after simultaneous rupture of the diaphragma and spleen, is a very rare condition. Most of the patients are asymptomatic and the lesions are detected accidentally. If the diagnosis can be made preoperatively, surgical excision is not needed.     

Cell Orientation Influences the Biaxial Mechanical Properties of Fibroblast Populated Collagen Vessels

Bioartificial tissues, composed of cells in a collagen matrix, can be fabricated with preferred cell orientations to mimic the histologic arrangement of biologic tissues. The influence of preferred cell orientations on the biaxial mechanical behavior of bioartificial tissues is unknown. Characterizing the biaxial mechanical behavior is necessary for better predicting the in vivo behavior of bioartificial tissues. Fibroblast populated collagen vessels (FPCVs) were fabricated with two different cell orientations by controlling the mechanical constraints during incubation. The cell orientation was verified by confocal microscopy and the collagen fiber organization was examined by confocal reflection and scanning electron microscopy (SEM). Pressure-diameter, force-length tests were performed to determine the influence of cell orientation on the biaxial mechanical behavior. FPCVs were more extensible in the direction perpendicular to the preferred cell orientation, than in the direction parallel to the cell orientation. Biaxial tests were also performed in the presence of Cytochalasin D (Cyto D) to minimize the mechanical contribution of the cells. After Cyto D treatment, the FPCVs remained more extensible in the direction perpendicular to the cell orientation, even though a preferred collagen fiber orientation was not observed in the microscopy images.     

Disseminated malignant solitary fibrous tumor of the pleura

Solitary fibrous tumor (SFT) of the pleura typically forms a localized pleura-based mass, and most are benign. A rare case of disseminated malignant SFT of the pleura is reported. The patient was a 71-year-old man who presented with complaints of shortness of breath to his primary care physician. A diagnosis of malignant mesothelioma was suspected, based on clinical, radiological and needle biopsy findings. He was referred to our institution for surgery. An extrapleural pneumonectomy, encompassing all pleural masses, was performed. Gross examination of the resected specimen was remarkable for numerous masses, ranging in size from 0.2 to 13.5 cm, covering the majority of the visceral pleura. Histologically, the tumor was composed of short spindle cells admixed with variable proportions of collagenous stroma. There were great intra- and intertumoral heterogeneity in tumor growth pattern, cellularity, pleomorphism and mitoses. Histologically malignant areas were present in all of the masses examined. The neoplastic cells were diffusely and intensely positive for bcl-2. Most tumor cells were also strongly stained for CD34 and CD99. Staining for cytokeratin was negative. The tumor also revealed p53 over-expression. Thus, the histological and immunohistochemical features of the tumor were consistent with a disseminated malignant SFT. This report shows that SFT rarely presents with disseminated pleural involvement, and a panel with CD34, bcl-2 and cytokeratin are valuable for differentiating SFT from malignant mesothelioma and other malignant spindle cell neoplasms of the pleura.     

Ultrastructural observations on the histogenesis of localized fibrous tumours of the pleura (benign mesothehoma)

Summary Five localized fibrous tumours of the pleura (benign mesothelioma) were studied ultrastructurally in order to elucidate their histogenesis. The histological subtypes of this benign fibrous lesion of the visceral pleura, i.e. the cellular, the collagenous, and the hyaline, were separately analysed.The tumours are composed of undifferentiated mesenchymal cells, intermediate and differentiated fibroblasts as well as collagenous interstitial tissue. The varying distribution of these cell elements account for the various histological subtypes. Morphological similarities between the mesenchymal tumour cells and the superficial mesothelial cells, which are always separated from the true tumour tissue by an intact basement membrane, were not observed.The different cellular elements can be regarded as parts of a continuous spectrum of cytodifferentiation, in which the mature fibroblasts are derived via intermediate forms from the undifferentiated cells. It is concluded that the localized fibrous tumours of the pleura arise from immature mesenchymal stem cells, which seems to be normally found in the submesothelial layer of the visceral pleura.     

Na+-glucose cotransporter is also expressed in mesothelium of species with thick visceral pleura

Molecular evidence for Na+-glucose cotransporter (SGLT1) in rabbit pleural mesothelium has been recently provided, confirming earlier functional findings on solute-coupled liquid absorption from rabbit pleural space. In this research we checked whether SGLT1 is also expressed in pleural mesothelium of species with thick visceral pleura, which receives blood from systemic circulation, but drains it into pulmonary veins. To this end immunoblot assays were performed on total protein extract of scraped visceral and parietal mesothelium of lambs and adult sheep, and of a human mesothelial cell line. All of them showed SGLT1 specific bands. Moreover, confocal immunofluorescence images of lamb pleural mesothelium showed that SGLT1 is located in apical membrane. Therefore, a solute-coupled liquid absorption should also occur from pleural space of species with thick visceral pleura. Because of this protein-free liquid entering interstitium between visceral mesothelium and capillaries, inherent Starling forces should be different than hitherto considered, and visceral pleura capillaries could absorb liquid even in these species.     

Isolated pleural PTLD after cardiac transplantation

Preamble: Epstein–Barr virus infection (EBV) and immunosuppression promote emergence of posttransplant lymphoproliferative disorders (PTLD) in patients undergoing organ transplantation. Objective: We report a case of PTLD confined to the pleura. Findings: The patient was a 62-year-old male who had undergone cardiac transplant in 1993 for ischemic heart disease. Seven years later, he presented with dyspnea and bilateral pleural effusions. The CT scan revealed left sided pleural base thickening. The cytology of the pleural fluid and fine needle aspirate of the pleura was both suggestive of PTLD. However, the tissue submitted for ancillary studies did not contain the diagnostic material. A clinical decision was made to withdraw immunosuppressive therapy and start rituximab. His clinical course was complicated by Pneumocystis carinii pneumonia and he died 4 months after the diagnosis of PTLD. Autopsy revealed bilateral pleural effusions with pleural nodules involving the visceral and parietal pleura of both lungs. Immunohistochemistry demonstrated B cell lineage with κ/λ ratio of 1. PCR studies done on the pleural nodules (postmortem specimen) revealed the presence of EBV DNA and absence of human herpes virus 8 (HHV8) DNA. In situ hybridization revealed positive staining for EBV RNA within the neoplasm. Conclusion: Pleural-based PTLD is rare. Cytology in conjunction with immunophenotyping and molecular studies can be useful for a definitive diagnosis. In our case, cytology sample was suggestive of PTLD. PCR studies performed on the antemortem specimen confirmed the presence of monoclonal IgH gene rearrangement, while the postmortem specimen revealed oligoclonal IgH gene rearrangement. The change from monoclonal to oligoclonal IgH gene rearrangement suggests reversion of monoclonal to polyclonal PTLD following rituximab and CHOP therapy. We also demonstrated EBV DNA and RNA in the tumor nodules, supporting EBV-induced PTLD.     

The significance of pleural elastica invasion by lung carcinomas.

Invasion of visceral pleura by primary lung carcinomas is an important parameter in staging. The complex histology of visceral pleura requires special elastic stains for proper evaluation, yet only approximately 10% of peripheral lung carcinomas seen in consultation (S.J.U.) are thus assessed. The objective of this study was to examine the prognostic importance of microscopic visceral pleura invasion by lung carcinomas. Retrospective analysis of 23 cases of peripheral T2, N0, M0 carcinomas with microscopic pleural invasion on elastic stains and a matched control group documented a statistically significant (P = 0.0236) difference in survival between squamous cell carcinoma subgroups. This study therefore suggests the need for histologic assessment of peripheral lung carcinomas for invasion of internal pleural elastic lamina.     

Biodegradation of polyurethane under fatigue loading

A method utilizing expansion of a diaphragm-type film specimen was developed to study in vitro biodegradation of poly(etherurethane urea) (PEUU) under conditions of dynamic loading (fatigue). A finite element model was used to describe the strain state, which ranged from uniaxial at the edges of the film to balanced biaxial tensile strain at the center. During testing, the film was exposed to a H(2)O(2)/CoCl(2) solution, which simulated in vivo oxidative biodegradation of PEUU. The extent of chemical degradation was determined by infrared analysis. Physical damage of the film surface was characterized by optical microscopy and scanning electron microscopy. Dynamic loading did not affect the rate of degradation relative to unstressed and constant stress (creep) controls in regions of the film that experienced primarily uniaxial fatigue; however, degradation was accelerated in regions that experienced balanced biaxial or almost balanced biaxial fatigue. It was concluded that the combination of dynamic loading and biaxial tensile strain accelerated oxidative degradation in this system. Chemical degradation produced a brittle surface layer that was marked by numerous pits and dimples. Physical damage of the surface in the form of cracking occurred only in fatigue experiments. Cracking was not observed in unstressed or creep tests. Cracks initiated at the dimples produced by chemical degradation, and propagated in a direction that was determined by the strain state.     

Structure,blood supply,and lymphatic vessels of the sheep's visceral pleura

We investigated the morphology of the visceral pleura of 36 sheep, using macroscopic, histologic, and ultrastructural approaches to quantify regional pleural thickness, blood supply, and lymphatic drainage, including the pulmonary ligament and hilar lymphatic distributions. Pleural thickness increased caudally and dorsally, such that the costal pleura of the caudal lobes had a mean minimum pleural thickness of 83 μm. The blood supply to the entire visceral pleura came exclusively from the bronchial arteries. Lymph vessels formed an extensive plexus throughout the serous membrane of all lobes. Trunk lymphatics (> 100 μm diameter) had a density of about 2/cm of pleural length on all lobar surfaces except for the cranial and middle lobes, where their density on the costal surfaces was ≤ 1/cm. Pleural trunk lymphatics coursed to the pulmonary ligaments and to the hilum on their way to regional lymph nodes. At the hilum they anastomose with intrapulmonary lymphatic trunks. The principal lymph nodes to receive pulmonary lymph were the caudal mediastinal node and tracheobronchial nodes. The visceral pleura of sheep is thick, showing considerable regional diversity in morphology.     

A constitutive model of the posterior cruciate ligament

Further to our previous work on the development of a general constitutive framework for transversely isotropic viscohyperelasticity (Limbert, G, Middleton, J. A transversely isotropic viscohyperelastic material. Application to the modelling of biological soft connective tissues. Int J Solids Struct 2004;41(15):4237-60.), we propose a phenomenological constitutive law to describe the anisotropic viscohyperelastic behaviour of the human posterior cruciate ligament (PCL) at high strain rates. The mechanical formulation is based on the definition of a Helmholtz free energy function containing a hyperelastic and a viscous potential. The equations are valid for arbitrary kinematics and satisfy elemental thermodynamic principles. Identification of the constitutive model with experimental data obtained from human PCL specimens was performed and showed the ability of the model to capture accurately the mechanical characteristics of the PCL at various strain rates. Influence of the isotropic and directional viscous stress responses on the global mechanical response are discussed in connection with the modelling hypotheses. This work was motivated by the need to provide an accurate constitutive model of the PCL to be used in finite element analyses of human occupants in car crash simulations. Besides uniaxial tests along the natural fibre orientation of the PCL, additional tests such as equibiaxial, strip biaxial compression-tension and shear tests were also performed in order to assess the physical response of the model in different loading situations. It was found that the model performed as well in these conditions.