La aplicación secuencial de cubreobjetos de vidrio para evaluar la compresión Rigidez de la lente del ratón: tensión y el análisis morfométricos
Summary
Age-related increases in eye lens stiffness are linked to presbyopia. This protocol describes a simple, cost-effective method for measuring mouse lens stiffness. Mouse lenses, like human lenses, become stiffer with age. This method is precise and can be adapted for lenses from larger animals.
Abstract
El lente del ojo es un órgano transparente que refracta y enfoca la luz para formar una imagen nítida en la retina. En los seres humanos, los músculos ciliares contrato para deformar la lente, lo que lleva a un aumento de la potencia óptica de la lente para enfocar objetos cercanos, un proceso conocido como alojamiento. cambios relacionados con la edad en la rigidez de la lente se han vinculado a la presbicia, una reducción en la capacidad de la lente 'para acomodar, y, por extensión, la necesidad de gafas de lectura. A pesar de que las lentes de ratón no se acomodan o desarrollar la presbicia, modelos de ratón pueden proporcionar una herramienta invaluable para patologías genéticas de lentes de comprensión, y el envejecimiento acelerado observado en ratones permite el estudio de los cambios relacionados con la edad en la lente. Este protocolo se muestra un método simple, preciso y rentable para la determinación de la rigidez de la lente ratón, utilizando cubreobjetos de vidrio para aplicar secuencialmente creciente cargas de compresión sobre la lente. Representante de datos confirman que las lentes de ratón se vuelven más rígidos con la edad, comolentes humanos. Este método es altamente reproducible y potencialmente se puede escalar hasta mecánicamente lentes de ensayo de los animales más grandes.
Introduction
The lens is a transparent and avascular organ in the anterior chamber of the eye that is responsible for fine focusing of light onto the retina. A clear basement membrane, called the lens capsule, surrounds a bulk of elongated fiber cells covered by an anterior monolayer of epithelial cells1,2. Life-long growth of the lens depends on the continuous proliferation and differentiation of epithelial cells at the lens equator into new fiber cells that are added onto previous generations of fiber cells in a concentric manner2. Over time, lens fiber cells undergo compaction, resulting in a rigid center in the middle of the lens called the nucleus1. Accommodation, defined as a dioptric change in the optical power of the eye, occurs in humans when the ciliary muscles contract to deform the lens and allow a true increase in optical power to focus on near objects3-5. In the unaccommodated eye, the lens is held in a relatively flattened state due to tension from zonular fibers. When the ciliary muscles contract, the tension on the lens is released, leading to decreased lens equatorial diameter and increased axial thickness. Age-related changes in the lens cause presbyopia, a progressive loss of lens accommodation, which leads to the need for reading glasses.
Several studies have linked presbyopia to age-related increase in the intrinsic stiffness of the lens6-11. Stiffness is defined as the resistance of an elastic object to deform under applied load. A variety of methods have been used to examine stiffness of human lenses, including spin compression12-14, actuator compression15, probe indentation16, dynamic mechanical analysis 6,10 and bubble-based acoustic radiation force17. While mouse lenses do not accommodate or develop presbyopia, mouse models for lens pathologies are valuable tools because mice are less expensive than larger animals, well characterized genetically and undergo accelerated age-related changes due to rapid aging. A handful of studies have examined mouse lens stiffness with compression methods and demonstrated changes in lens stiffness due to aging or targeted genetic disruptions18-21. Thus, mouse lenses are good models for studying age-related changes in lens stiffness.
This protocol describes a simple and inexpensive, yet precise and reproducible, compression method for determining mouse lens stiffness based on application of glass coverslips onto the lens in conjunction with photographing the lens through a dissection microscope and mirror. This method yields robust strain and morphometric data with an easily fabricated and assembled apparatus. The representative results confirm that mouse lenses increase in stiffness with age.
Protocol
Representative Results
Discussion
Hay varias consideraciones clave al utilizar este método para medir la rigidez de la lente. En primer lugar, los cubreobjetos se aplican a la lente en un ángulo ligeramente oblicuo (8-8,5 °) con respecto a la parte inferior de la cámara (θ). Esto se aplicará un componente muy pequeño de la carga ecuatorial en lugar de axialmente. Sin embargo, esta carga ecuatorial se considera insignificante porque el pecado θ ≈ 0,1 19. Si este método se adapta para lentes más grandes, tendría que ser medida para…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
This work was supported by National Eye Institute Grant R01 EY017724 (VMF) and National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant K99 AR066534 (DSG).
Materials
| Fine tip straight forceps | Fine Scientific Tools | 11252-40 | |
| Microdissection scissors, straight edge | Fine Scientific Tools | 15000-00 | |
| Curved forceps | Fine Scientific Tools | 11272-40 | |
| Seizing forceps | Hammacher | HSC 702-93 | Optional |
| Dissection dish | Fisher Scientific | 12565154 | |
| 60mm petri dish | Fisher Scientific | 0875713A | |
| 1X phosphate buffered saline (PBS) | Life Technologies | 14190 | |
| 18mm x 18mm glass coverslips | Fisher Scientific | 12-542A | |
| Measurement chamber with divots to hold lenses | Custom-made (see Figure 1) | ||
| Right-angle mirror | Edmund Optics | 45-591 | |
| Light source | Schott/Fostec | 8375 | |
| Illuminated dissecting microscope | Olympus | SZX-ILLD100 | With SZ-PT phototube |
| Digital camera | Nikon | Coolpix 990 |
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