Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract

Summary

Cataract is the leading cause of blindness in the world. Solar ultraviolet radiation (UVR) is the main risk factor for cataract development. An animal model of far UVR-B induced cataract was developed. In this article we describe methods for investigation of cataract formation: exposure to UVR, quantitative RT-PCR and immunohistochemistry.

Abstract

Cataract is the leading cause of blindness in the world ¹. The World Health Organization defines cataract as a clouding of the lens of the eye which impedes the transfer of light. Cataract is a multi-factorial disease associated with diabetes, smoking, ultraviolet radiation (UVR), alcohol, ionizing radiation, steroids and hypertension. There is strong experimental ^2-4 and epidemiological evidence ^5,6 that UVR causes cataract. We developed an animal model for UVR B induced cataract in both anesthetized ⁷ and non-anesthetized animals ⁸.

The only cure for cataract is surgery but this treatment is not accessible to all. It has been estimated that a delay of onset of cataract for 10 years could reduce the need for cataract surgery by 50% ⁹. To delay the incidence of cataract, it is needed to understand the mechanisms of cataract formation and find effective prevention strategies. Among the mechanisms for cataract development, apoptosis plays a crucial role in initiation of cataract in humans and animals ¹⁰. Our focus has recently been apoptosis in the lens as the mechanism for cataract development ^8,11,12. It is anticipated that a better understanding of the effect of UVR on the apoptosis pathway will provide possibilities for discovery of new pharmaceuticals to prevent cataract.

In this article, we describe how cataract can be experimentally induced by in vivo exposure to UVR-B. Further RT-PCR and immunohistochemistry are presented as tools to study molecular mechanisms of UVR-B induced cataract.

Protocol

1. Exposure to Ultraviolet Radiation

1. 15 min before the exposure, anesthetize a female Sprague-Dawley rat with a mixture of 90 mg/kg Ketalar (ketamine) and 10 mg/kg Rompun (xylazine) by intraperitoneal injection.
2. Place the animal in a rat restrainer and tighten the belts until immobilization of the rat without causing a trunk squeeze ¹³.
3. Instill Mydriacyl (tropicamide), 10 mg/ml, in both eyes of the rat to induce mydriasis.
4. Place the animal so that one eye is positioned against a narrow beam of UVR at 300 nm ¹⁴ with 10 nm full width at half maximum and shield the contralateral eye with black tape.

Results

The various sources of variation in the measurements were estimated with an analysis of variance and it was found that considering three measurements per animal the variance for measurements was on the order of 15 % of that for animals. Thus, considering whole lens analysis, it is not possible to increase the precision. Orthogonal testing elucidated a statistically significant contrast for caspase-3 message between 120 hr latency interval versus shorter latency intervals.

In vivo UVR .......

Discussion

This paper describes methods that enable studying molecular events occurring during UVR-B induced cataract.

Considering, that most information available for in vivo UVR induced cataract was derived from experiments on albino Sprague-Dawley rats ^{7, 16, 17, 18, 19}, we decided to use the albino Sprague-Dawley rat in the current study. Age of the rats was six week old. The gender was chosen to be female because, in contrast to males, females have less allergenic urine. Moreover.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Name of the reagent	Company	Catalogue number	Comments (optional)
Ketalar	Pfizer	150086	50 mg/ml
Rompun	Bayer	022545	20 mg/ml
Oculentum Simplex	Apoteket, Sweden	336164	5 g
Mydriacyl	Alcon	00352	10 mg/ml
Balanced salt solution	Alcon	0007950055
3.5 ul β-mercaptoethanol
NucleoSpin RNA II total RNA isolation kit	Macherey-Nagel GmbH&Co, Duren, Germany	740955.50
p53 DNA specific primers	biomers.net GmbH	Custom made
Taq DNA polymerase, dNTPack	Roche	04 728 866 001
Agarose	Sigma	A5093
Ethidium bromide solution 0.5 mg/ml	Sigma	E1385
Nano-Drop ND-1000 spectrophotometer	NanoDrop Products
1st Strand cDNA synthesis kit for RT-PCR (AMV)	Roche Diagnostics GmbH	11 483 188 001
iCycler MyiQ Single Color Real Time PCR detection system	Bio-Rad Laboratories
96-well plate	Sarstedt	72.1979.202
TaqMan Gene Expression Master Mix	Applied Biosystems	4369016
TaqMan Gene Expression Assay for caspase 3	Applied Biosystems	Rn00563902_m1
TaqMan Gene Expression Assay for 18s	Applied Biosystems	Hs99999901_s1
MyiQ software	Bio-Rad Laboratories
Cleaved caspase-3 (Asp175)	Cell signaling technology	9661
Anti rabbit IgG	Abcam	Ab6798
Sucrose	Sigma Aldrich	84097
Vectashield	Vector Laboratories
Universal Microscope Axioplan 2 Imaging	Carl Zeiss
Paraformaldehyde	Sigma Aldrich	441244
TBE buffer 10x	Promega	V4251