A gimbal-mounted pressurization chamber for macroscopic and microscopic assessment of ocular tissues

The biomechanical model of glaucoma considers intraocular pressure-related stress and resultant strain on load bearing connective tissues of the optic nerve and surrounding peripapillary sclera as one major causative influence that effects cellular, vascular, and axonal components of the optic nerve. By this reasoning, the quantification of variations in the microstructural architecture and macromechanical response of scleral shells in glaucomatous compared to healthy populations provides an insight into any variations that exist between patient populations. While scleral shells have been tested mechanically in planar and pressure-inflation scenarios the link between the macroscopic biomechanical response and the underlying microstructure has not been determined to date. A potential roadblock to determining how the microstructure changes based on pressure is the ability to mount the spherical scleral shells in a method that does not induce unwanted stresses to the samples (for instance, in the flattening of the spherical specimens), and then capturing macroscopic and microscopic changes under pressure. Often what is done is a macroscopic test followed by sample fixation and then imaging to determine microstructural organization. We introduce a novel device and method, which allows spherical samples to be pressurized and macroscopic and microstructural behavior quantified on fully hydrated ocular specimens. The samples are pressurized and a series of markers on the surface of the sclera imaged from several different perspectives and reconstructed between pressure points to allow for mapping of nonhomogenous strain. Pictures are taken from different perspectives through the use of mounting the pressurization scheme in a gimbal that allows for positioning the sample in several different spherical coordinate system configurations. This ability to move the sclera in space about the center of the globe, coupled with an upright multiphoton microscope, allows for collecting collagen, and elastin signal in a rapid automated fashion so the entire globe can be imaged.     

Multiscale modeling of calcium cycling in cardiac ventricular myocyte: macroscopic consequences of microscopic dyadic function

In cardiac ventricular myocytes, calcium (Ca) release occurs at distinct structures (dyads) along t-tubules, where L-type Ca channels (LCCs) appose sarcoplasmic reticulum (SR) Ca release channels (RyR2s). We developed a model of the cardiac ventricular myocyte that simulates local stochastic Ca release processes. At the local Ca release level, the model reproduces Ca spark properties. At the whole-cell level, the model reproduces the action potential, Ca currents, and Ca transients. Changes in microscopic dyadic properties (e.g., during detubulation in heart failure) affect whole-cell behavior in complex ways, which we investigated by simulating changes in the dyadic volume and number of LCCs/RyR2s in the dyad, and effects of calsequestrin (CSQN) as a Ca buffer (CSQN buffer) or a luminal Ca sensor (CSQN regulator). We obtained the following results: 1), Increased dyadic volume and reduced LCCs/RyR2s decrease excitation-contraction coupling gain and cause asynchrony of SR Ca release, and interdyad coupling partially compensates for the reduced synchrony. 2), Impaired CSQN buffer depresses Ca transients without affecting the synchrony of SR Ca release. 3), When CSQN regulator function is impaired, interdyad coupling augments diastolic Ca release activity to form Ca waves and long-lasting Ca release events.     

Long range intermolecular forces between macroscopic bodies: macroscopic and microscopic approaches.

Van der Waals energies of interaction are calculated by two methods, the macroscopic method of Lifshitz and the microscopic method of London-Casimir and Polder-Hamaker for the case of two semi-infinite slabs separated by a thin film. When retardation effects may be neglected, the London-Hamaker approach yields values of dispersion interactions which almost coincide with those of the Lifshitz approach, the magnitude of the former values being larger by approximately 10–25%, which is attributed to the effect of the molecular environment in condensed media. At 50–100 Å film thicknesses where retardation effects are small, dispersion terms are generally the major part of van der Waals forces in the Lifshitz formulation. Hence, for 50–100 Å film thicknesses the Hamaker approach, which only includes dispersion interactions is generally adequate. By accounting for retardation effects, which significantly reduce the magnitude of dispersion interactions at several hundred Å, there is a reasonable agreement between the values obtained by the macroscopic and microscopic approaches. When polar substances are present and for film thicknesses of several hundred Å, where dispersion interactions are significantly reduced, the major contribution to van der Waals forces may arise from orientation and induction terms. For such cases the Hamaker approach may lead to critical underestimates of the calculated magnitude of van der Waals forces. An ad hoc way to overcome this difficulty which is applicable to any geometry is proposed. This study presents a simple procedure for the determination of free energies of interaction between macroscopic bodies of various shapes. The procedure, which is applicable when the molecules of bodies and surrounding medium are isotropic, yields results which closely approximate those obtained with the Lifshitz theory.     

Species of the Bacillus strain: macroscopic and microscopic morphology

Between October 2001 and January 2002, the Microbiology Group of the Instituto Nacional de Salud processed 705 envelopes under suspicion of harboring anthrax spores. We present photographs of cultures and slides prepared from them of Bacillus species to be used as reference material for the accurate macroscopic and microscopic identification of the agent found in samples.     

Exploring a biological tissue from atomic to macroscopic scale using synchrotron radiation: example of hair.

A combined approach, using synchrotron radiation-based diffraction and infrared microspectrometry, has been used to study the structure and molecular composition of hair samples. These methods allowed us to get an insight at different structural scales into the composition and structure of hair. Firstly, information about the configuration of amino-acid residues was obtained at atomic scale, secondly, a model was presented for the geometry and the packing of the microfibrils at medium scale and finally different structural zones were evidenced by microdiffraction at macroscopic scale. We also showed that the two main components of hair--proteins and lipids--are not evenly distributed within the fiber. In addition, these two components exhibit different structure, depending upon their location. Moreover, diffraction and microdiffraction data indicate that the cuticle zone is mainly composed of lipid granules, whereas the cortex and the medulla zones are composed primarily of alpha-keratin. Infrared microspectroscopy, using an enhanced lateral resolution thanks to synchrotron radiation, indicates, on one hand, that the protein structure between the cuticle and cortex are different, and on the other hand, that the concentration of lipids, inside the medulla, is much higher than everywhere else. This work emphasizes the complementarity between both techniques, and highlights the potentialities they can offer in the case of various other studies in biology.     

From macroscopic measurements to microscopic mechanisms of protein aggregation

The ability to relate bulk experimental measurements of amyloid formation to the microscopic assembly processes that underlie protein aggregation is critical in order to achieve a quantitative understanding of this complex phenomenon. In this review, we focus on the insights from classical and modern theories of linear growth phenomena and discuss how theory allows the roles of growth and nucleation processes to be defined through the analysis of experimental in vitro time courses of amyloid formation. Moreover, we discuss the specific signatures in the time course of the reactions that correspond to the actions of primary and secondary nucleation processes, and outline strategies for identifying and characterising the nature of the dominant process responsible in each case for the generation of new aggregates. We highlight the power of a global analysis of experimental time courses acquired under different conditions, and discuss how such an analysis allows a rigorous connection to be established between the macroscopic measurements and the rates of the individual microscopic processes that underlie the phenomenon of amyloid formation.     

Linking microscopic spatial patterns of tissue destruction in emphysema to macroscopic decline in stiffness using a 3D computational model

Pulmonary emphysema is a connective tissue disease characterized by the progressive destruction of alveolar walls leading to airspace enlargement and decreased elastic recoil of the lung. However, the relationship between microscopic tissue structure and decline in stiffness of the lung is not well understood. In this study, we developed a 3D computational model of lung tissue in which a pre-strained cuboidal block of tissue was represented by a tessellation of space filling polyhedra, with each polyhedral unit-cell representing an alveolus. Destruction of alveolar walls was mimicked by eliminating faces that separate two polyhedral either randomly or in a spatially correlated manner, in which the highest force bearing walls were removed at each step. Simulations were carried out to establish a link between the geometries that emerged and the rate of decline in bulk modulus of the tissue block. The spatially correlated process set up by the force-based destruction lead to a significantly faster rate of decline in bulk modulus accompanied by highly heterogeneous structures than the random destruction pattern. Using the Karhunen-Loève transformation, an estimator of the change in bulk modulus from the first four moments of airspace cell volumes was setup. Simulations were then obtained for tissue destruction with different idealized alveolar geometry, levels of pre-strain, linear and nonlinear elasticity assumptions for alveolar walls and also mixed destruction patterns where both random and force-based destruction occurs simultaneously. In all these cases, the change in bulk modulus from cell volumes was accurately estimated. We conclude that microscopic structural changes in emphysema and the associated decline in tissue stiffness are linked by the spatial pattern of the destruction process.     

Electron microscopic analysis of lymphocyte fibrillar elements during the redistribution of surface receptors

Filaments 5 nm thick, located throughout the cytoplasm mainly along the surface, are observed in intact lymphocytes. In the control glycerinized lymphocytes, besides the above filaments aggregations of filaments nearly 3 nm in diameter were found. After the treatment of cells by antimurine serum or ferritin-conjugated concanavalin A, some fibrillar structures are observed mainly in the cap region in the form of filaments 5-6 nm of thickness, radially directed towards the plasma membrane. After glycerinization, three types of filaments are observed being, respectively, near 3, 5-6 and almost 8 nm in diameter. Two latter types of filaments are decorated by S1-myosine fragments which indicates their actine nature. Differences in the character and distribution of myofibrils in the cytoplasm of intact cells and cells with caps may witness in favour of their involvement in the processes associated with redistribution of surface receptors.     

Effects of microscopic and macroscopic viscosity on the rate of renaturation of DNA

The effect of solvent viscosity on DNA renaturation rates has been investigated as a function of temperature for a number of solvent systems. The results are all consistent with a microscopic viscosity limitation of the rate determining step. Rates of renaturation in perchlorate and quaternary ammonium salt solutions are also discussed. Increasing the macroscopic viscosity with dissolved neutral or anionic polymers increases, rather than decreases, renaturation rates due to the excluded volume of the dissolved polymers.     

Application of the macroscopic electric charge balance in fermentation modeling

This article treats the application of the macroscopic electric charge balance in fermentation modeling. From the presented calculations it follows that the definition formula of the so-called degree of reduction is changed due to the ionic character of the fermentation reactants. It is also shown that the macroscopic electric charge balance, together with the ionic equilibria between fermentation reactants, leads to an expression for the calculation of the pH during fermentation. Finally it is concluded that one should be very careful in the estimation of biomass production from the acid or base feed rates which are necessary for pH control.     

Comparative macroscopic and microscopic anatomical observations on pyloric ceca in some bony fish

The authors report the variable number of pyloric caeca of some species of fish and describe their macroanatomy. They also describe and compare the microanatomy of pyloric caeca in three species; in particular they point out the thickness of tunica muscularis, the shape and length of villi which protrude into the lumen and the variable number of goblet cells. The tunica muscularis is very strong in Uranoscopus and consists of three strata: external and inner longitudinal strata, middle circular stratum. The tunica muscularis is less thick in Diplodus and in Scorpaena. The lamina propria and epithelium form villi which protrude into the lumen. They are covered with columnar epithelium and goblet cells which are more numerous in Uranoscopus and Scorpaena and less numerous in Diplodus. At last the authors describe in pyloric caeca of Scorpaena the presence of a network which engages all the lumen; it consists of connective tissue septa which directly derive from the lamina propria and is covered with the same columnar epithelium and goblet cells.     

Ethanol effect on the chick embryo ossification: a macroscopic and microscopic study

This study attempts to analyze anomalies in avian embryos induced macroscopically and microscopically when exposed to ethanol (EtOH) during the first stages of development. Fertilized chicken eggs were employed in this study. The eggs were incubated at 37.8 degrees C. Some of the eggs were treated on day 0 with EtOH (20%, 40% and 60%) by instillation in the air sac. The control group was instilled with 0.1 ml of NaCl at 0.9%. Other eggs were treated on the 4th post-incubation day, employing the same methodology. The embryos in both groups were removed from the eggs on the 11th incubation day and examined using a dissecting binocular microscope. After macroscopic analysis, the samples obtained were fixed in 10% formol, photographed and processed according to common histological techniques and the Picrosirius method. Embryos treated with EtOH demonstrated a significant weight decrease. Microscopic analysis by means of the Picrosirius method revealed that the intra-membranous ossification process presents less development, and therefore there was less type I collagen in trabecular bone in the embryos post-exposure to EtOH with respect to the control.     

Utrastructural interrelationships of collagen fibrillar components in human connective tissue]

The data on structural interconnections between collagenous fibres in the human dermis and periosteum were obtained by means of raster electron microscopy. Collagenous fibres were demonstrated to be connected with each other by means of fine connective fibres situating mainly transversaly towards the main collagenous fibres. Comparing the data obtained with those from the literature, a suggestion was made on the existence, in the connective tissue, of a special connective system composing of peculiar fibrillar structures which maintains dynamic equilibrium in arrangement of collagenous fibres, muscles, vessels, etc. The system of the connective fibres demonstrates a common compositional principle for all the structures mentioned above, but in every case having its peculiarities.     

Long-term remodeling of vascularized and nonvascularized onlay bone grafts: a macroscopic and microscopic analysis

The present study was performed to compare vascularized and nonvascularized onlay bone grafts to investigate the potential effect of graft-to-recipient bed orientation on long-term bone remodeling and changes in thickness and microarchitectural patterns of remodeling within the bone grafts. In two groups of 10 rabbits each, bone grafts were raised bilaterally from the supraorbital processes and placed subperiosteally on the zygomatic arch. The bone grafts were oriented parallel to the zygomatic arch on one side and perpendicular to the arch on the contralateral side. In the first group, vascularized bone grafts were transferred based on the auricularis anterior muscle, and in the second group nonvascularized bone grafts were transferred. Fluorochrome markers were injected during the last 3 months of animal survival, and animals were killed either 6 or 12 months postoperatively. The nonvascularized augmented zygoma showed no significant change in thickness 6 months after bone graft placement and a significant decrease in thickness 1 year after graft placement (p < 0.01). The vascularized augmented zygoma showed a slight but statistically significant decrease in thickness 6 months after graft placement (p < 0.003), with no significant difference relative to its initial thickness 1 year after graft placement. In animals killed 6 months after bone graft placement, both the rate of remodeling and the bone deposition rate measured during the last 3 months of survival were significantly higher in the vascularized bone grafts compared with their nonvascularized counterparts (p < 0.02). By 1 year postoperatively, there were no significant differences in thickness, mineral apposition rate, or osteon density between bone grafts oriented perpendicular and parallel to the zygomatic arch. These findings indicate that the vascularity of a bone graft has a significant effect on long-term thickness and histomorphometric parameters of bone remodeling, whereas the direction of placement of a subperiosteal graft relative to the recipient bed has minimal effect on these parameters. In vascularized bone grafts, both bone remodeling and deposition are accelerated during the initial period following graft placement. Continued bone deposition renders vascularized grafts better suited for the long-term maintenance of thickness and contour relative to nonvascularized grafts.     

Light microscopic immunolocation of thrombospondin in human tissues

Affinity-purified antisera against thrombospondin were used to locate the presence of this glycoprotein in frozen sections of several human tissues by immunofluorescence techniques. Immunostaining was observed in the peritubular connective tissue and in basement membrane regions beneath glandular epithelium in skin and lung. Intense immunostaining was observed at the dermal-epidermal junction in skin and in small blood vessels throughout this tissue. Skeletal muscle exhibited positive staining with anti-thrombospondin antisera within interstitial areas. Immunostaining was confined to the luminal portions of large blood vessels such as aorta. In large blood vessels that contained lesions of atherosclerosis, immunostaining was observed throughout the lesion area and was especially prominent surrounding some of the lesion cells. These results indicate that thrombospondin is located within the matrix of a variety of human tissues and supports the suggestion that this glycoprotein is an endogenous component of some extracellular matrices.