마우스 렌즈의 압축 강성을 평가하는 유리 Coverslips는 순차적으로 응용 프로그램 : 스트레인과 형태 학적 분석
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
눈 렌즈는 굴절 및 망막에 선명한 화상을 형성하는 광을 집중 투명 기관이다. 인간, 모양체 근육 계약은 가까운 물체에 초점 렌즈 '광 파워, 숙박 시설로 알려진 공정의 증가로 이어지는, 렌즈를 변형합니다. 렌즈 강성 연령 관련 변경 사항이 노안에 연결되었습니다 확장하여 수용 할 수있는 렌즈의 능력 감소하고, 안경을 독서의 필요성. 마우스 렌즈 수용 노안을 개발하지 않더라도, 마우스 모델 이해 렌즈 병리위한 귀중한 유전 적 수단을 제공 할 수 있으며, 마우스에서 관찰 된 가속 열화 렌즈의 연령과 관련된 변화에 대한 연구를 가능하게한다. 이 프로토콜은, 마우스 렌즈 강도를 결정 순차적으로 적용되는 렌즈에 압축 하중을 증가시키는 커버 글라스를 사용하는 단순하고 정확하고 비용 효율적인 방법을 설명한다. 대표적인 데이터는 마우스 렌즈처럼 나이가 엄격한되고 있는지 확인인간의 렌즈. 이 방법은 높은 재현성과 잠재적으로 더 큰 동물 시험 렌즈를 기계적으로까지 확장 할 수 있습니다.
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
렌즈의 강도를 측정하는이 방법을 사용할 때 몇 가지 중요한 고려 사항이 있습니다. 챔버 (θ)의 저부에 대하여 – (8.5 ° 8) 우선, 커버 슬립 약간 비스듬한 각도로 렌즈에 적용된다. 이 적도에 축보다는 오히려 부하의 매우 작은 구성 요소를 적용한다. 그러나이 적도 부하가 죄 θ ≈ 0.1 (19) 때문에 무시할 간주됩니다. 이 방법은 큰 렌즈에 적합한 경우, 상기 챔버의 하부에 커버 슬립 각도 적?…
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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