マウスレンズの圧縮剛性を評価するために、ガラスカバースリップの逐次適用：ひずみと形態計測分析
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
眼のレンズは屈折し、網膜上に鮮明な画像を形成するために光を集光する透明な器官です。ヒトでは、毛様体筋の契約は、近くの物体に焦点を合わせるレンズの光学的パワー、宿泊として知られているプロセスの増加につながる、レンズを変形させます。加齢に関連するレンズ剛性の変化老眼にリンクされている、対応するレンズの能力の減少、および、拡張によって、老眼鏡が必要とされています。マウスのレンズは老眼を受け入れるか、発症しないにもかかわらず、マウスモデルが理解レンズ病理のための貴重な遺伝的なツールを提供することができ、およびマウスで観察された促進老化は、レンズ内の加齢変化の研究を可能にします。このプロトコルは、レンズに圧縮荷重を増加させる、順次適用し、ガラスカバースリップを使用して、マウスレンズ剛性を決定するために、簡単、正確、かつ費用効果的な方法を示します。代表的なデータは、マウスのレンズのような、年齢とともにより硬くなっていることを確認します人間のレンズ。この方法は、非常に再現性があり、潜在的により大きな動物から試験レンズを機械的にスケールアップすることができます。
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
レンズの剛性を測定するために、このメソッドを使用する際には、いくつかの重要な考慮事項があります。室（θ）の底面に対して – （8.5°8）まず、カバーガラスを少し斜めの角度でレンズに適用されます。これは非常に小さな負荷のコンポーネント赤道ではなく、軸方向に適用されます。罪θ≈0.1 19しかし、この赤道負荷は無視できると考えられています。この方法は、より大きな?…
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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