Recent investigations of human corneal structure and biomechanics have shown that stromal collagen fibers (lamellae) are organized into a complex, highly intertwined three-dimensional meshwork of ...transverse oriented fibers that increases stromal stiffness and controls corneal shape. The purpose of this study was to characterize the three-dimensional distribution of transverse collagen fibers along the major meridians of the cornea using an automated method to rapidly quantify the collagen fibers' angular orientation.
Three eyes from three donors were perfusion-fixed under pressure, excised, and cut into four quadrants. Quadrants were physically sectioned using a vibratome and scanned using nonlinear optical high-resolution macroscopy. Planes were analyzed numerically using software to identify collagen fiber angles relative to the corneal surface, stromal depth, and radial position within the anterior 250 μm of the stroma.
The range of fiber angles and the fiber percentage having an angular displacement greater than ±3.5° relative to the corneal surface ("transverse fibers") was highest in the anterior stroma and decreased with depth. Numerical analysis showed no significant differences in fiber angles and transverse fibers between quadrants, meridians, and radial position.
These results match our previous observation of a depth-dependent gradient in stromal collagen interconnectivity in the central cornea, and show that this gradient extends from the central cornea to the limbus. The lack of a preferred distribution of angled fibers with regard to corneal quadrant or radial position likely serves to evenly distribute loads and to avoid the formation of areas of stress concentration.
Outcome of Repeat Pulmonary Metastasectomy Krüger, Marcus; Franzke, Katharina; Rajab, Taufiek Konrad ...
Advances in experimental medicine and biology,
01/2021, Letnik:
1335
Journal Article
Recenzirano
Pulmonary metastasectomy is a well-established contribution to the cure of oligometastatic cancers, but its exact effectiveness is poorly understood. Here we report the outcomes of repeat pulmonary ...metastasectomy from a multicenter trial. This retrospective study included patients who underwent re-do metastasectomies between January 2010 and December 2014. The exclusion criterion was metastasectomy without curative intent. We reviewed medical files of 621 consecutive patients who underwent initial pulmonary metastasectomy. Of those, 64 patients underwent repeat metastasectomies, and these patients were included in the analysis. All the 64 patients underwent a second metastasectomy, later 35 of them underwent a third metastasectomy, 12 underwent a fourth metastasectomy, and 6 underwent a fifth metastasectomy. The total number of re-do metastasectomies was 181. The median overall survival among the patients undergoing re-do metastasectomy was 66.0 ± 3.8 months. Three and 5-year survival rates were 82.3% and 63.3%, respectively. The 5-year survival rates were 63.3% after the first, 50.9% after the second, 74.4% after the third, 83.3% after the fourth, and 60.0% after the fifth metastasectomy. We conclude that at the current stage of knowledge, there is an indication for repeat re-do metastasectomy with curative intent.
The cornea is the most external layer of the eye and serves two important roles in (1) the refraction of light and (2) protection from the outside environment, both of which are highly dependent on ...the collagen assembly of the corneal stroma. This study sought to determine the collagen fiber arrangement of the canine corneal stroma and correlate the stromal organization with tissue stiffness in the anterior and posterior cornea. Collagen organization of the canine cornea was visualized through second-harmonic generation (SHG) imaging, and tissue stiffness of the anterior and posterior corneal stroma was determined by atomic force microscopy. Analysis of the canine anterior corneal stroma using SHG imaging documented intertwining of the collagen fibers with a high degree of fiber branching, with a more lamellar and non-branching posterior stroma. The anterior stroma had significantly higher tissue stiffness in both dogs and humans, when compared with the posterior corneal stroma (canine median: 1.3 kPa vs. 0.3 kPa; human median: 14.6 kPa vs. 2.1 kPa, respectively). There was a direct correlation between corneal collagen stromal organization and tissue stiffness in the dog, which was consistent with other mammalian species previously examined and likely reflects the need for maintenance of rigidity and corneal curvature.
Recent developments in nonlinear optical (NLO) imaging using femtosecond lasers provides a noninvasive method for detecting collagen fibers by imaging second harmonic-generated (SHG) signals. ...However, this technique is limited by the small field of view necessary to generate SHG signals. The purpose of this report is to review our efforts to greatly extend the field of view to assess the entire collagen structure using high-resolution macroscopic (HRMac) imaging.
Intact human eyes were fixed under pressure, and the whole cornea (13-mm diameter) was excised and embedded in low-melting point agar for vibratome sectioning (200-300 microm). Sections were then optically scanned using a Zeiss LSM 510 Meta and Chameleon femtosecond laser (Carl Zeiss Microimaging Inc., Thornwood, NY) to generate SHG images. For each vibratome section, an overlapping series of three-dimensional data sets (466 x 466 x 150 microm) were taken, covering the entire tissue (15 mm x 6 mm area) using a motorized, mechanical stage. The three-dimensional data sets were then concatenated to generate an NLO-based tomograph.
The HRMac of the cornea yielded large macroscopic (80 megapixels per plane), three-dimensional tomographs with high resolution (0.81 microm lateral, 2.0 microm axial) in which individual collagen fibers (stromal lamellae) could be traced, segmented, and extracted. Three-dimensional reconstructions suggested that the anterior cornea comprises highly intertwined lamellae that insert into the anterior limiting lamina (Bowman's layer).
We conclude that HRMac using NLO-based tomography provides a powerful new tool to assess collagen structural organization within the cornea.
As the primary structural protein of our bodies, fibrillar collagen and its organizational patterns determine the biomechanics and shape of tissues. While the molecular assembly of individual fibrils ...is well understood, the mechanisms determining the arrangement of fibers and thus the shape and form of tissues remain largely unknown. We have developed a cell culture model that successfully recapitulates early tissue development and the
de novo
deposition of collagen fibers to investigate the role of mechanical cues on collagen fiber alignment. The devices used a thin, collagen-coated deformable PDMS membrane inside a tissue culture well built on microscope-grade coverslips. Deformations and strains in the PDMS membrane were quantified by tracking fluorescent bead displacement and through the use of a COMSOL model. Cyclical strains were applied to serum-cultured rabbit corneal cells at 0.5 Hz for 24–48 h and showed a preferred alignment after 36 h of cyclical loading. Cells cultured with ascorbic acid under methylcellulose serum-free conditions deposited a collagenous matrix that was visible under live second harmonic generation microscopy at week 4. Our microfabricated tissue culture system allows for the controllable application of a wide range of stress profiles to cells, and for the observation and quantification of cells and de novo collagen formation
in vitro
. Future studies will involve the fabrication of models to study the formation and organization of collagen in ocular diseases.
Although the cornea is the major refractive element of the eye, the mechanisms controlling corneal shape and hence visual acuity remain unknown. To begin to address this question we have used ...multiphoton, non-linear optical microscopy to image second harmonic generated signals (SHG) from collagen to characterize the evolutionary and structural changes that occur in the collagen architecture of the corneal stroma. Our studies show that there is a progression in complexity of the stromal collagen organization from lower (fish and amphibians) to higher (birds and mammals) vertebrates, leading to increasing tissue stiffness that may control shape. In boney and cartilaginous fish, the cornea is composed of orthogonally arranged, rotating collagen sheets that extend from limbus to limbus with little or no interaction between adjacent sheets, a structural paradigm analogous to 'plywood'. In amphibians and reptiles, these sheets are broken down into broader lamellae that begin to show branching and anastomosing with adjacent lamellae, albeit maintaining their orthogonal, rotational organization. This paradigm is most complex in birds, which show the highest degree of lamellar branching and anastomosing, forming a 'chicken wire' like pattern most prominent in the midstroma. Mammals, on the other hand, diverged from the orthogonal, rotational organization and developed a random lamellar pattern with branching and anastomosing appearing highest in the anterior stroma, associated with higher mechanical stiffness compared to the posterior stroma.
To characterize and quantify the collagen fiber (lamellar) organization of human corneas in three dimensions by using nonlinear optical high-resolution macroscopy (NLO-HRMac) and to correlate these ...findings with mechanical data obtained by indentation testing of corneal flaps.
Twelve corneas from 10 donors were studied. Vibratome sections, 200 μm thick, from five donor eyes were cut along the vertical meridian from limbus to limbus (arc length, 12 mm). Backscattered second harmonic-generated (SHG) NLO signals from these sections were collected as a series of overlapping 3-D images, which were concatenated to form a single 3-D mosaic (pixel resolution: 0.44 μm lateral, 2 μm axial). Collagen fiber intertwining was quantified by determining branching point density as a function of stromal depth. Mechanical testing was performed on corneal flaps from seven additional eyes. Corneas were cut into three layers (anterior, middle, and posterior) using a femtosecond surgical laser system and underwent indentation testing to determine the elastic modulus for each layer.
The 3-D reconstructions revealed complex collagen fiber branching patterns in the anterior cornea, with fibers extending from the anterior limiting lamina (ALL, Bowman's layer), intertwining with deeper fibers and reinserting back to the ALL, forming bow spring-like structures. Measured branching-point density was four times higher in the anterior third of the cornea than in the posterior third and decreased logarithmically with increasing distance from the ALL. Indentation testing showed an eightfold increase in elastic modulus in the anterior stroma.
The axial gradient in lamellar intertwining appears to be associated with an axial gradient in the effective elastic modulus of the cornea, suggesting that collagen fiber intertwining and formation of bow spring-like structures provide structural support similar to cross-beams in bridges and large-scale structures. Future studies are necessary to determine the role of radial and axial structural-mechanical heterogeneity in controlling corneal shape and in the development of keratoconus, astigmatism, and other refractive errors.
The rabbit is commonly used to evaluate new corneal prosthetics and study corneal wound healing. Knowledge of the stiffness of the rabbit cornea would better inform the design and fabrication of ...keratoprosthetics and substrates with relevant mechanical properties for in vitro investigations of corneal cellular behavior. This study determined the elastic modulus of the rabbit corneal epithelium, anterior basement membrane (ABM), anterior and posterior stroma, Descemet’s membrane (DM) and endothelium using atomic force microscopy (AFM). In addition, three-dimensional collagen fiber organization of the rabbit cornea was determined using nonlinear optical high-resolution macroscopy. The elastic modulus as determined by AFM for each corneal layer was: epithelium, 0.57±0.29kPa (mean±SD); ABM, 4.5±1.2kPa, anterior stroma, 1.1±0.6kPa; posterior stroma, 0.38±0.22kPa; DM, 11.7±7.4kPa; and endothelium, 4.1±1.7kPa. The biophysical properties, including the elastic modulus, are unique for each layer of the rabbit cornea and are dramatically softer in comparison to the corresponding regions of the human cornea. Collagen fiber organization is also dramatically different between the two species, with markedly less intertwining observed in the rabbit vs. human cornea. Given that the substratum stiffness considerably alters the corneal cell behavior, keratoprosthetics that incorporate mechanical properties simulating the native human cornea may not elicit optimal cellular performance in rabbit corneas that have dramatically different elastic moduli. These data should allow for the design of substrates that better mimic the biomechanical properties of the corneal cellular environment.
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