盖玻片序贯应用评估鼠标透镜的抗压刚度：应变和形态分析
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
眼透镜是透明的器官，折射和光聚焦，以形成在视网膜上的清晰图像。在人类中，睫状肌收缩变形的透镜，从而增加在透镜“光功率集中于附近的物体，被称为住宿的方法。在镜头的刚度与年龄相关的变化都与老花眼，在适应镜头的能力降低，以及由此延伸，需要老花镜。尽管鼠标镜头不适合或开发老花眼，小鼠模型可以为理解镜头病症的宝贵基因工具，并在小鼠中观察到加速老化能够在镜头年龄相关的变化研究。这个协议表明用于确定鼠标透镜刚度，用玻璃盖玻片依次施加压缩负荷增大到透镜的简单，精确和成本效益的方法。有代表性的数据证实，鼠标透镜随着年龄的增长变得更硬，像人类的镜头。这种方法是高度可重复的，并且可以潜在地扩大到从较大的动物机械地测试镜片。
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
使用这种方法来衡量镜片硬度时，有几个关键因素。 （ – 8.5°8）相对于所述腔室（θ）的底部第一，盖玻片在略微倾斜的角度施加到透镜。这将适用于负载的非常小的组分平展，而不是轴向。然而，这种赤道负载可以忽略不计，因为罪θ≈0.1 19。如果此方法适于大透镜，盖玻片到腔室的底部的角将需要被测量以确定赤道负载是否应计入应变计算。其次，它是重要的，以允许透镜到加?…
Disclosures
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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