Abstract
purpose. The purpose of the present study was to investigate metabolic changes in different compartments of the rat lens (anterior, nuclear, posterior, and equatorial) after exposure to an acute double threshold dose of ultraviolet-B radiation (UVR-B) by using high-resolution magic angle spinning (HR-MAS) 1H nuclear magnetic resonance (NMR) spectroscopy and pattern recognition (PR) methods.
methods. One eye in each of 28 6-week-old female albino Sprague-Dawley rats was exposed to in vivo 7.5 kJ/m2 UVR-B for 15 minutes. The contralateral eye was left unexposed. One week after irradiation, all rats were killed, and both lenses were isolated. Each lens was cored by a trephine, and the cylinder was sliced into three portions (anterior, nuclear, and posterior). The lens material that remained after the coring process was analyzed as the equatorial region. Analysis of lens metabolism was performed by HR-MAS 1H NMR spectroscopy (14.1 T; Avance DRX600; Bruker BioSpin GmbH, Rheinstetten, Germany), and the metabolic profiles were statistically analyzed by the PR method of principal component analysis (PCA).
results. Metabolic differences were detected among the compartments in the lens, both in samples from the contralateral nonexposed lenses and in samples from lenses exposed to in vivo UVR-B. In the rat lens, exposure to UVR-B caused changes in GSH, phosphocholine, myo-inositol, succinate, formate, and adenosine triphosphate (ATP)/adenosine diphosphate (ADP) and in levels of the amino acids phenylalanine, taurine, hypo-taurine, tyrosine, alanine, valine, isoleucine, and glutamate, that varied among lens compartments.
conclusions. HR-MAS 1H NMR spectroscopy, combined with PR methods (PCA), is effective for analysis of separate parts of the intact rat lens. To understand the biochemistry of the lens, it is important to divide the lens into sections, representing functionally and anatomically distinct compartments.
Exposure to UVR-B (280−315 nm) is thought to be a major cause of cataract, and because of the thinning of the ozone layer, an increase in solar UVR-B is of current concern. The ozone layer prevents normally 70% to 90% of the UVR-B to reach the earth’s surface, and the incoming radiation varies considerably with geographic, physical, and meteorological factors.
1 When UVR-B at 300-nm reaches the eye, the cornea, and the aqueous humor attenuates 97% of the incident radiation.
2 The remaining 3% is completely attenuated in the lens.
2 Evidence for cataractogenesis caused by the high-energy wavelengths of UVR-B is found both in animal experiments and in human studies.
3
The effects of UVR-B on the lens differ between acute high-dose and chronic low-dose exposure.
4 Acute high-dose exposure to UVR-B causes cortical opacities
5 and induces several changes in the lens, such as alterations in enzyme activity caused by an increased amount of free radicals and inactivation of free radical scavenging systems.
6 Electrolyte transport problems
7 and DNA damage
8 9 are associated with osmotic changes in the lens, leading to cortical opacification. Chronic low-dose exposure to UVR-B affects tryptophan and may form crystalline links, which cause sclerosis and opacities in the nucleus of the lens.
4
The pathogenic effects of UVR-B vary among lens compartments. Over the course of a lifetime, lens fiber cells differentiate to form different parts of the lens. The cortical region is histologically different from the nuclear area. The epithelial cells at the equator of the lens proliferate and transform into new fiber cells. These cells further develop as the cortical area and thereafter as the nuclear part of the lens. The nucleus consists of the oldest and deepest lens fiber cells, which have lost all organelles. Exposure to previous UVR-B, is suggested to be a reason for nuclear opacity.
5 In contrast, opacities observed in the cortical area, consisting of relatively newly developed fiber cells, might be a result of recent UVR-B exposure.
5
In the investigation of photochemical damage of the lens, studies of metabolism are helpful in understanding the underlying processes. In previous studies of metabolism, substantial differences have been observed between different compartments of the lens.
10 In studies of the biochemical properties of the whole lens, only the average of the ingredient substances have been analyzed.
10 11 Data on the topography of biochemical alterations in the lens are limited. Separate analyses of different lenticular compartments will increase the understanding of pathophysiology in the lens.
High-resolution magic angle spinning (HR-MAS)
1H nuclear magnetic resonance (NMR) spectroscopy of metabolic changes in different compartments of the intact rat lens can be investigated with quality as good as from extracts.
11 NMR-based metabonomics is a modern but well-known statistical method of handling the large amount of data from NMR spectra.
12 These methods have only recently been applied to the eye.
13 14 15 16 HR-MAS
1H NMR spectroscopy has revealed considerable changes in the rat lens biochemistry after exposure to UVR-B.
11 A significant decrease was found in several amino acids such as valine, phenylalanine, tyrosine, taurine, hypo-taurine, glycine, and glutamate.
11 However, these results presented just the average of the ingredient substances in the whole lens.
In the present study, we investigated the biochemical response alterations in the various compartments of the albino rat lens after in vivo exposure to UVR-B. This is the first study to present such detailed biochemical data on intact tissue from different sections of the lens. Because of histologic differences in the lens, we hypothesized that the metabolic response of UVR-B exposure would be different between the lens compartments. A detailed biochemical analysis using 1H HR-MAS NMR spectroscopy may contribute to a better understanding of the mechanisms by which UVR-B induces cataractogenesis in various parts of the lens.
Six-week-old female outbred albino Sprague-Dawley rats (n = 28, ∼150 g) were anesthetized with 11 mg/kg xylazine and 80 mg/kg ketamine, intraperitoneally. Before irradiation, 1 drop of 0.5% tropicamide was instilled in both eyes to dilate the pupils. One eye of each animal was exposed to UVR-B and the contralateral eye served as a nonexposed control. The UVR-B dose was 7.5 kJ/m2, measured in the corneal plane, and the exposure time was 15 minutes. The UVR-B source was a 350-W high-pressure Hg lamp (Oriel Instruments, Stratford, CT) equipped with water filter, dual monochromator set to λMAX at 300 nm and full bandwidth at half maximum (FWHM) 10 nm, and collimating optics. The irradiance was measured with a thermopile calibrated by the Swedish National Bureau of Standards. The maximum intensity was at 300.1 nm, and true FWHM was 9.5 nm. All animals were kept and treated according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
One week after exposure, the rats were killed by CO
2 asphyxiation, the eyes were enucleated, and the lenses were placed in physiologic saline (BSS; Alcon, Stockholm, Sweden) and dissected from the remnants of ciliary body and zonular fibers. The lenses were stored at −20°C for 30 to 60 minutes before compartmentalization of the frozen lenses. A Bonn Sectioning Device (Department of Experimental Ophthalmology, University of Bonn, Germany)
17 was cooled by circulating methanol from a water bath kept at −5°C. All accessories used for the compartmentalization was cooled by submersion in liquid nitrogen. Each rat lens was cored with a trephine (2.5 mm bore) and sliced into three regions. The lens core was divided into a 1-mm anterior portion and a 1-mm nuclear portion, and the remaining posterior portion had a thickness varying from 0.7 to 1.5 mm. The lens material left after the coring process was analyzed as the equatorial region. Samples for NMR analysis were made from 28 exposed lenses and 28 control lenses, where each NMR sample consisted of pooled equivalent parts from 4 different lenses. The average weight (± SD) of the pooled samples were 22.0 ± 2.9 (anterior), 22.6 ± 1.7 (nucleus), 19.3 ± 2.8 (posterior), and 19.6 ± 3.5 (equatorial) mg. Two NMR samples were discarded because of destruction in the laboratory. Immediately after sectioning, the pooled lens fractions were stored at −80°C.
NMR spectra were statistically analyzed by principal component analysis (PCA; The Unscrambler ver. 7.01; CAMO, Trondheim, Norway). PCA is a common statistical technique for finding patterns in data with multiple response variables. It reduces dimensionality and produces new uncorrelated variables (principal components), describing the amount of variation in the data set. A selected region (0.65–4.7 ppm) from the HR-MAS 1H NMR spectra (54 samples), was used as input for PCA. This area included the most abundant metabolites in a single spectrum of the rat lens. The dominating resonances of phosphocholine (3.16−3.21, 3.47−3.51 and 4.02− 4.07 ppm) were removed from the selected regions. In this way, potential differences between the compounds of lower intensities were easier to reveal. The low-field region (above 5 ppm) had a lower signal-to-noise ratio and was not included in the analysis. The results are based on a matrix consisting of 54 samples × 25,380 variables. PCA was performed with full cross-validation, indicating that the same samples were used for both calibration and validation. The number of principal components (PCs) included in the cross-validation analysis was determined by the explained variance. The number of PCs that gave the minimal total residual variance was considered to be the required number of PCs. To find group divisions and reveal the relationship between samples, the score plot from the various PCs was interpreted.
Amino Acids.
Antioxidants.
Metabolism.
Energy Transfer.
Methyl Group Donors and Membrane Building Blocks.
Cell Signaling and Osmoregulation.