In situ cryofixation of lung tissue using the PS1000, a hand-held metal mirror cryofixation device

1992 ◽  
Vol 50 (1) ◽  
pp. 740-741
Author(s):  
John J. Godleski ◽  
Rebecca C. Stearns ◽  
Marshall Katler ◽  
Robyn Rufner ◽  
Theresa D. Sweeney ◽  
...  
Keyword(s):  
Lung Tissue ◽  
Internal Surface ◽  
Lung Parenchyma ◽  
Natural State ◽  
Phagocytic Cells ◽  
Inhaled Particles ◽  
Large Airways ◽  
Distal Lung

We have previously reported methods to cryofix the extracellular lining layers of the large airways and trachea. We seek to study the extracellular lining layers of both airways and alveoli in situ because it is the interaction of these lining materials with inhaled particles that determines the subsequent responses of phagocytic cells to the particles. Cryopreservation of the lining fluids of lung parenchyma is difficult because of the complexity of distal lung structure, and the need to fix the lung in its natural state, i.e., filled with air. Any method used must be able to obtain the frozen specimen from the inflated lung. This requirement precludes the possibility of having a cryogen come into primary contact with the internal surface of an alveolus. However, if optimal fixation through the pleura could be attained, then the lining layers of subjacent alveoli could be studied. In this report, we describe a new method to cryopreserve distal lung tissue for optimal study of the extracellular lining layers of the alveolus. Alveolar tissue is sufficiently preserved in the inflated state to maintain its in vivo characteristics.

scholarly journals Penetration of Meropenem into Pneumonic Human Lung Tissue as Measured by In Vivo Microdialysis

2004 ◽  
Vol 48 (6) ◽  
pp. 2228-2232 ◽  
Author(s):  
Florian Tomaselli ◽  
Alfred Maier ◽  
Veronika Matzi ◽  
Freyja Maria Smolle-Jüttner ◽  
Peter Dittrich
Keyword(s):  
Lung Tissue ◽  
Human Lung ◽  
Pleural Empyema ◽  
Lung Parenchyma ◽  
In Vivo Microdialysis ◽  
Human Lung Tissue ◽  
Distribution Profile ◽  
Significant Difference ◽  
Pharmacokinetic Properties

ABSTRACT Until recently, information on antibiotic pharmacokinetic properties in infected human lung tissue was limited. We therefore studied a microdialysis-based approach for measurement of the penetration of meropenem into the extracellular space fluid of human pneumonic lung parenchyma. The lung penetration of meropenem was determined for seven patients with pneumonia and metapneumonic pleural empyema treated by decortication. Intraoperatively, two microdialysis probes were inserted into pneumonic lung tissue and one was inserted into healthy skeletal muscle for reference values. Serum and microdialysis samples were collected at 20-min intervals for at least 8 h following a single intravenous dose of 1 g of meropenem. The maximum free interstitial concentration (mean and standard deviation) of meropenem in infected lung tissue was 11.4 ± 10.9 mg/liter, and that in serum was 47.3 ± 21.0 mg/liter. The areas under the curve for infected lung tissue (36.2 ± 17.9 mg · h/liter) and serum (95.4 ± 46.6 mg · h/liter) revealed a significant difference. This technique enabled quasi-continuous tissue pharmacokinetic measurements of free, unbound antibiotic in pneumonic lung tissue of patients with pneumonia. The present data corroborate the use of meropenem in the treatment of lung infections caused by extracellular bacteria, demonstrating the excellent distribution profile for meropenem in the interstitial space of human pneumonic lung tissue.

Biaxial distension of precision-cut lung slices

2010 ◽  
Vol 108 (3) ◽  
pp. 713-721 ◽  
Author(s):  
C. Dassow ◽  
L. Wiechert ◽  
C. Martin ◽  
S. Schumann ◽  
G. Müller-Newen ◽  
...  
Keyword(s):  
Lung Tissue ◽  
Cell Injury ◽  
Mechanical Stretch ◽  
Longitudinal Direction ◽  
Lung Parenchyma ◽  
Cell Types ◽  
Static Conditions ◽  
Precision Cut Lung Slices

The mechanical forces acting on lung parenchyma during (mechanical) ventilation and its (patho)physiological consequences are currently under intense scrutiny. Several in vivo and cell culture models have been developed to study the pulmonary responses to mechanical stretch. While providing extremely useful information, these models do also suffer from limitations in being either too complex for detailed mechanical or mechanistic studies, or in being devoid of the full complexity present in vivo (e.g., different cell types and interstitial matrix). Therefore in the present study it was our aim to develop a new model, based on the biaxial stretching of precision-cut lung slices (PCLS). Single PCLS were mounted on a thin and flexible carrier membrane of polydimethylsiloxane (PDMS) in a bioreactor, and the membrane was stretched by applying varying pressures under static conditions. Distension of the membrane-PCLS construct was modeled via finite element simulation. According to this analysis, lung tissue was stretched by up to 38% in the latitudinal and by up to 44% in the longitudinal direction, resulting in alveolar distension similar to what has been described in intact lungs. Stretch for 5 min led to increased cellular calcium levels. Lung slices were stretched dynamically with a frequency of 15/min for 4 h without causing cell injury {3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) test; live/dead straining}. These findings suggest that stretching of PCLS on PDMS-membranes may represent a useful model to investigate lung stretch in intact lung tissue in vitro for several hours.

scholarly journals Optical Detection of Distal Lung Enzyme Activity in Human Inflammatory Lung Disease

BME Frontiers ◽  
2021 ◽  
Vol 2021 ◽  
pp. 1-11
Author(s):  
Alicia Megia-Fernandez ◽  
Adam Marshall ◽  
Ahsan R. Akram ◽  
Bethany Mills ◽  
Sunay V. Chankeshwara ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Lung Disease ◽  
Drug Target ◽  
Human Lung ◽  
Matrix Metalloproteases ◽  
Therapeutic Drug ◽  
Experimental Medicine ◽  
Distal Lung

Objective and Impact Statement. There is a need to develop platforms delineating inflammatory biology of the distal human lung. We describe a platform technology approach to detect in situ enzyme activity and observe drug inhibition in the distal human lung using a combination of matrix metalloproteinase (MMP) optical reporters, fibered confocal fluorescence microscopy (FCFM), and a bespoke delivery device. Introduction. The development of new therapeutic agents is hindered by the lack of in vivo in situ experimental methodologies that can rapidly evaluate the biological activity or drug-target engagement in patients. Methods. We optimised a novel highly quenched optical molecular reporter of enzyme activity (FIB One) and developed a translational pathway for in-human assessment. Results. We demonstrate the specificity for matrix metalloproteases (MMPs) 2, 9, and 13 and probe dequenching within physiological levels of MMPs and feasibility of imaging within whole lung models in preclinical settings. Subsequently, in a first-in-human exploratory experimental medicine study of patients with fibroproliferative lung disease, we demonstrate, through FCFM, the MMP activity in the alveolar space measured through FIB One fluorescence increase (with pharmacological inhibition). Conclusion. This translational in situ approach enables a new methodology to demonstrate active drug target effects of the distal lung and consequently may inform therapeutic drug development pathways.

scholarly journals In Situ Metal Mirror Cryofixation Using a Portable, Hand-Held Instrument

1992 ◽  
Vol 00 (4) ◽  
pp. 4-4
Keyword(s):  
Proximal Tubule ◽  
Pleural Fluid ◽  
Materials Science ◽  
Rat Kidney ◽  
Clinical Pathology ◽  
Lung Parenchyma ◽  
Biological Research ◽  
Natural State

With an increasing interest in both immunological procedures and elemental (ion) detection, it is imperative that tissues be preserved in as life-like a state as possible. Up until now the only type instrument available for in situ cryo fixation was various configurations of cryopliers which lacked the ability to control freezing variables. With the introduction of the PS1000 (Delaware Diamond Knives, Inc.), a pneumatically operated hand held slammer, it is now possible to reproducibly fix in situ as well as in vitro and achieve results seen previously only with table top instruments. This instrument is designed to provide a consistent delivery rate through the use of a pneumatically operated piston.The availability of such an instrument, clearly improves the accuracy of many investigations. Pleural fluid of the lung parenchyma (Figure 1) has been preserved in its natural state (i.e., filled with air). Figure 2 shows a proximal tubule of rat kidney which has been crycgenically preserved in situ. Applications in biological research, materials science and clinical pathology are currently being investigated at several major institutions.

In vitro and in vivo polysaccharides assembly: ultrastructural and cytochemical study of growing plant cell wall components.

1978 ◽  
Vol 36 (2) ◽  
pp. 434-435
Author(s):  
D. Reis ◽  
B. Vian ◽  
J. C. Roland
Keyword(s):  
Cell Wall ◽  
Self Assembly ◽  
Plant Cell Wall ◽  
Higher Plants ◽  
Three Dimensional ◽  
Cytochemical Study ◽  
Wall Components

Wall morphogenesis in higher plants is a problem still open to controversy. Until now the possibility of a transmembrane control and the involvement of microtubules were mostly envisaged. Self-assembly processes have been observed in the case of walls of Chlamydomonas and bacteria. Spontaneous gelling interactions between xanthan and galactomannan from Ceratonia have been analyzed very recently. The present work provides indications that some processes of spontaneous aggregation could occur in higher plants during the formation and expansion of cell wall.Observations were performed on hypocotyl of mung bean (Phaseolus aureus) for which growth characteristics and wall composition have been previously defined.In situ, the walls of actively growing cells (primary walls) show an ordered three-dimensional organization (fig. 1). The wall is typically polylamellate with multifibrillar layers alternately transverse and longitudinal. Between these layers intermediate strata exist in which the orientation of microfibrils progressively rotates. Thus a progressive change in the morphogenetic activity occurs.

In situ microprobe quantitation of inorganic particle burden in paraffin sections of lung tissue

1988 ◽  
Vol 46 ◽  
pp. 990-991
Author(s):  
Jerrold L. Abraham
Keyword(s):  
Lung Tissue ◽  
Quantitative Information ◽  
Particulate Material ◽  
Paraffin Sections ◽  
Tissue Samples ◽  
Qualitative And Quantitative ◽  
The Past ◽  
Quantitative Analyses ◽  
Data Result

Inorganic particulate material of diverse types is present in the ambient and occupational environment, and exposure to such materials is a well recognized cause of some lung disease. To investigate the interaction of inhaled inorganic particulates with the lung it is necessary to obtain quantitative information on the particulate burden of lung tissue in a wide variety of situations. The vast majority of diagnostic and experimental tissue samples (biopsies and autopsies) are fixed with formaldehyde solutions, dehydrated with organic solvents and embedded in paraffin wax. Over the past 16 years, I have attempted to obtain maximal analytical use of such tissue with minimal preparative steps. Unique diagnostic and research data result from both qualitative and quantitative analyses of sections. Most of the data has been related to inhaled inorganic particulates in lungs, but the basic methods are applicable to any tissues. The preparations are primarily designed for SEM use, but they are stable for storage and transport to other laboratories and several other instruments (e.g., for SIMS techniques).

Three-Dimensional Subcellular Organization of Hepatocytes in Intact Liver: Simultaneous Visualization of Cytoskeletal and Membrane Markers by Confocal Microscopy

1996 ◽  
Vol 54 ◽  
pp. 288-289
Author(s):  
Greg V. Martin ◽  
Ann L. Hubbard
Keyword(s):  
Three Dimensional ◽  
Integral Membrane Proteins ◽  
Polarized Secretion ◽  
Microscope Images ◽  
Computer Aided ◽  
Intracellular Organelles ◽  
Cytoskeletal Elements ◽  
Simultaneous Visualization

The microtubule (MT) cytoskeleton is necessary for many of the polarized functions of hepatocytes. Among the functions dependent on the MT-based cytoskeleton are polarized secretion of proteins, delivery of endocytosed material to lysosomes, and transcytosis of integral plasma membrane (PM) proteins. Although microtubules have been shown to be crucial to the establishment and maintenance of functional and structural polarization in the hepatocyte, little is known about the architecture of the hepatocyte MT cytoskeleton in vivo, particularly with regard to its relationship to PM domains and membranous organelles. Using an in situ extraction technique that preserves both microtubules and cellular membranes, we have developed a protocol for immunofluorescent co-localization of cytoskeletal elements and integral membrane proteins within 20 µm cryosections of fixed rat liver. Computer-aided 3D reconstruction of multi-spectral confocal microscope images was used to visualize the spatial relationships among the MT cytoskeleton, PM domains and intracellular organelles.

Biomedical applications: Pitfalls in the practice

1994 ◽  
Vol 52 ◽  
pp. 378-379
Author(s):  
J. D. Shelburne ◽  
Peter Ingram ◽  
Victor L. Roggli ◽  
Ann LeFurgey
Keyword(s):  
Operating Room ◽  
Liquid Nitrogen ◽  
Lung Tissue ◽  
Biomedical Applications ◽  
Microprobe Analysis ◽  
Tissue Sample ◽  
Cellular Level ◽  
Tissue Samples ◽  
Asbestos Fibers

At present most medical microprobe analysis is conducted on insoluble particulates such as asbestos fibers in lung tissue. Cryotechniques are not necessary for this type of specimen. Insoluble particulates can be processed conventionally. Nevertheless, it is important to emphasize that conventional processing is unacceptable for specimens in which electrolyte distributions in tissues are sought. It is necessary to flash-freeze in order to preserve the integrity of electrolyte distributions at the subcellular and cellular level. Ideally, biopsies should be flash-frozen in the operating room rather than being frozen several minutes later in a histology laboratory. Electrolytes will move during such a long delay. While flammable cryogens such as propane obviously cannot be used in an operating room, liquid nitrogen-cooled slam-freezing devices or guns may be permitted, and are the best way to achieve an artifact-free, accurate tissue sample which truly reflects the in vivo state. Unfortunately, the importance of cryofixation is often not understood. Investigators bring tissue samples fixed in glutaraldehyde to a microprobe laboratory with a request for microprobe analysis for electrolytes.

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