Magnetic resonance imaging (MRI) study of the water content and transport in rat lenses

Experimental Eye Research 113 (2013) 162e171

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Magnetic resonance imaging (MRI) study of the water content and transport in rat lenses Egor A. Dobretsov a, b, Olga A. Snytnikova a, b, Igor V. Koptyug a, b, Robert Kaptein b, c, Yuri P. Tsentalovich a, b, * a b c

International Tomography Center SB RAS, Institutskaya Str. 3A, 630090 Novosibirsk, Russia Novosibirsk State University, Pirogova Str. 2, 630090 Novosibirsk, Russia Bijvoet Center, Utrecht University, Utrecht, The Netherlands

a r t i c l e i n f o

a b s t r a c t

Article history: Received 3 June 2013 Accepted in revised form 7 June 2013 Available online 18 June 2013

NMR micro-imaging technique has been used for the measurement of the water content distribution in lenses of senescence-accelerated OXYS rats and age-matched Wistar rats, as well as for the study of water and phosphate transport in rat lenses. The water content in the lens cortex is significantly higher than in the nucleus; the spatial gradient of the water content becomes steeper with age. No difference in the water content distribution has been found between Wistar and OXYS rat lenses of matching ages, although cataract onset in the OXYS rat lens occurs much earlier due to the enhanced generation of reactive oxygen species associated with oxidative stress. This finding implies that cataract development does not lead to significant changes in water content distribution inside the lens. The water transport in rat lenses slows down with age, and in OXYS lenses it is somewhat faster than in lenses of Wistar rats, probably due to the compensatory response to oxidative stress. The application of 31P MRI for the monitoring of phosphate penetration into a lens has been performed for the first time. It is found that phosphate transport in a lens is significantly slower than that of water. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: lens cataract MRI water transport

1. Introduction The main function of a lens is to focus light onto the retina, so the lens must be transparent, flexible (to provide accommodation), and should have a high refractive index. To meet these requirements, nature developed a specific design. Except the outer layer of epithelial cells, the lens mostly consists of organelle-free fiber cells packed with specific proteins, crystallins (Graw, 1997; Andley, 2007), with minimal metabolic activity and no protein synthesis. The lens transparency is provided by a homogeneous protein distribution inside the cells and a small intercellular spacing compared to the wavelengths of visible light. A lens is one of the few organs in which the turnover of its major constituents e proteins e is very small. Under oxidative stress, the crystallins undergo post-translational modifications, which accumulate throughout the lifespan (Bloemendal et al., 2004). Oxidative damage causes crystallin coloration, insolubilization, aggregation and precipitation. Eventually, these processes lead to the formation of

* Corresponding author. International Tomography Center, Institutskaya Str. 3A, 630090 Novosibirsk, Russia. Tel.: þ7 383 3303136; fax: þ7 383 3331399. E-mail address: [email protected] (Y.P. Tsentalovich). 0014-4835/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.exer.2013.06.008

large protein aggregates able to scatter light; as a result, with aging the lens hardens and clouds. Many researchers believe that the development of presbyopia and cataract is an inherent part of the aging process: indeed, age is by far the biggest risk factor for these diseases (Foster et al., 2008). However, some people maintain clear vision up to a very old age, which argues in favor of a principal distinction between cataract development and lens aging. The major cause of cataractogenesis is oxidative stress (Truscott, 2005; Williams, 2006; Fletcher, 2010) e an imbalance between proand antioxidants inside the lens. Reduced glutathione (GSH) plays a key role in prevention of oxidation (Reddy and Giblin, 1984; Giblin, 2000). The synthesis of GSH as well as the reduction of oxidized glutathione GSSG occurs in the outer part of the lens or outside the lens. The inner cells of the lens are unable to maintain sufficient amounts of antioxidants (Mathias et al., 1997), and the level of GSH in the lens nucleus is determined by the rate of diffusion of small molecules through the lens: in the absence of a vascular system, the diffusion is the sole mechanism of GSH delivery inside the lens and the removal of oxidized species. Thus, the study of the age-related and cataract-related changes in the diffusion through the lens may shed light on the general mechanism of cataractogenesis. At present, the detailed mechanism of the transport of nutrients and antioxidants into the lens nucleus is unknown. Several models

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have been suggested to describe the circulation of water and nutrients through the lens, including passive diffusion and active diffusion driven by ion currents (fluid circulation model (Mathias and Rae, 1985, 2004; Mathias et al., 2007)) or by activity of the epithelial cell layer which moves metabolites inside the lens via gap junctions (Goodenough et al., 1980; FitzGerald and Goodenough, 1986; Goodenough, 1992). Unfortunately, from the available experimental data it is difficult to evaluate the contribution of each of these mechanisms to the total lens transport system (Donaldson et al., 2010; Beebe and Truscott, 2010). One of the important findings is the discovery of the internal diffusion barrier at the border between cortex and nucleus (Sweeney and Truscott, 1998), which forms in the human lens at middle age and hinders metabolite movement into and out of the lens core. The lens internal structure, metabolite distribution, and transport of water and solutes inside the lens have been studied by numerous methods, among which the magnetic resonance imaging (MRI) technique occupies a very important place. This non-invasive method has been successfully used for the visualization of the human eye structure in vivo (Lizak et al., 2000; Patz et al., 2007; Jones et al., 2007; Kasthurirangan et al., 2008, 2011), measurement of the protein distribution inside the lens and the spatial gradient of refractive index (GRIN) (Garner et al., 2001; Moffat and Pope, 2002b; Moffat et al., 2002; Jones and Pope, 2004; Jones et al., 2005, 2007; Kasthurirangan et al., 2008), diffusivity mapping of the lens (Wu et al., 1993; Moffat and Pope, 2002a; Vaghefi et al., 2009), and real-time measurements of water transport in the lens (Moffat et al., 1999; Vaghefi et al., 2011, 2012). It has been shown that the refractive index of lens tissue is proportional to the protein concentration. At the same time, the proton relaxation time T2 of water also depends on the protein concentration (Moffat and Pope, 2002b; Moffat et al., 2002; Jones and Pope, 2004) due to the fast exchange of water with NH and OH groups in proteins (Hills et al., 1989). Thus, T2-mapping of the lens allows for the quantitative measurements of the refractive index distribution (Jones and Pope, 2004; Jones et al., 2005, 2007; Kasthurirangan et al., 2008). Diffusivity mapping demonstrated that water movement inside the lens is highly anisotropic (Moffat and Pope, 2002a; Vaghefi et al., 2009): diffusion along the fiber cells is relatively free, while the movement between cells is restricted by the cell membranes. The diffusion in the lens nucleus is significantly slower than in the cortex (Moffat et al., 1999; Vaghefi et al., 2009); with aging, the diffusion rate in the cortex changes insignificantly, whereas in the nuclei of 80-yearold human lenses it is 3e4 times slower than in young lenses. The existence of the diffusion barrier between cortex and nucleus of the human lens has been confirmed (Moffat and Pope, 2002a; Vaghefi et al., 2012). The present work is aimed at an MRI study of water and protein distribution and water transport in rat lenses. Two rat strains have been used: Wistar and senescence-accelerated OXYS rats. The OXYS strain was developed at the Institute of Cytology and Genetics SB RAS from the Wistar stock by selection for their susceptibility to the cataractogenic effect of galactose (Solov’eva et al., 1975). Recent studies have shown that rats of the OXYS strain meet the main requirements for the model of senile cataract (Kolosova et al., 2003a, 2003b; Marsili et al., 2004). It was shown that the first signs of cataract in OXYS rats appear at the age of 1.5 months; to the age of 3 months 90% of animals are affected by the lens opacification, and at the age of 4e6 months the morbidity reaches 100% (Kolosova et al., 2003b; Rumyantseva et al., 2008). The mature cataract was detected in 90% of eyes of two-year-old OXYS rats. For comparison, the initial signs of cataract appear in the Wistar lenses after 6 months. Thus, the comparison of T2 maps and diffusion rates in the lenses of Wistar and OXYS rats of different ages will make it possible to search for correlations between physical properties of

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the lens, on the one side, and the lens age and cataract development, on the other. 2. Experimental part 2.1. Animals and materials All animals were kept and treated according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The rats were housed in groups of five animals per cage (57 cm  36 cm  20 cm) and kept under standard laboratory conditions (at 22  2  C, 60% relative humidity, natural light), provided with a standard rodent diet PK120-1 (Laboratorsnab, Russia), and given water ad libitum. Animals were sacrificed by CO2 asphyxiation, the eyes enucleated and whole lenses removed. Lenses were obtained from senescence-accelerated OXYS rats at 20 days, 3, 12 and 18 months of age and from age-matched Wistar rats. The lenses were placed immediately in artificial aqueous humor (AAH), a synthetic medium similar to the aqueous humor in the anterior chamber of the eye, and stored in this solution at 37  C before the experiments (typically, from 1 to 5 h). The AAH was made from NaCl 130 mM; KCl 5 mM; MgCl2 0.5 mM; CaCl2 1 mM; NaHCO3 10 mM; glucose 5 mM; buffered with 10 mM HEPES to pH 7.1. Phosphate buffer solution was prepared with hydrates of Na2HPO4 and NaH2PO4. Reagents were purchased from Sigmae Aldrich. D2O 99.9% was from Armar Chemicals (Switzerland). 2.2. Measurement of the water content in the lens nucleus and cortex The nucleus of the rat lens was separated from the cortex by coring lenses with a 2.5-mm home-made stainless steel borer followed by cutting off of approximately 0.5 mm from each end of the core. The procedure was performed with lenses taken from storage at 70  C and warmed up to 18  C for easier cutting, all tools were cooled down to 18  C, and all manipulations were performed in a cold room at 5  C. The nucleus and the cortex (the combined doughnut-shaped outer remainder of boring and the ends of the core) were weighed, vacuum-dried, and then weighed again. From the dry-to-wet weight ratios, the water contents in the nucleus and the cortex of each lens were determined. 2.3. Magnetic resonance imaging experiments All experiments were carried out on a Bruker Avance III 400 MHz NMR spectrometer equipped with micro-imaging accessories. The gradient system provides linear gradients of the magnetic field up to 151.73 G/cm independently in all three spatial directions. For 1H MRI, a 15 mm diameter 1H RF insert was used. For 31 P experiments, a 25 mm 1H/31P RF insert was utilized. The lenses were taken out of AAH just before measurements and placed onto a fluoroplastic support in a 10 mm outside diameter NMR tube. The tube was positioned vertically in the RF probe of the instrument in such a way that the center of the lens was as close as possible to the isocenter of the gradient system and the center of the RF coil. After the manual tuning and matching of the radiofrequency probe, an image of a projection of the sample on a vertical plane was acquired in order to determine the actual position of the lens. The frequency of the slice selection pulse of the imaging sequence was then adjusted to ensure that an axial imaging slice passed through the center of the lens. After that, an appropriate imaging experiment was started immediately. Unless stated otherwise, all measurements were performed at ambient temperature 25e28  C. T2 measurements were performed in a dry sample

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tube with a small piece of wet cotton wool placed at the top of the tube, while T1 and PD maps were obtained for lenses immersed in AAH solution. For the acquisition of proton T2 maps of water, a twodimensional single-slice Carr-Purcell-Meiboom-Gill (CPMG) sequence was used which was optimized to make the value of the echo time TE as short as possible. A slice of 1 mm thickness was imaged with the field of view ca. 1  1 cm2 and an image matrix of 128  64 pixels, which corresponds to the in-plane spatial resolution of ca. 78  154.5 mm2. The image slice was selected perpendicular to the vertical axis of the NMR sample tube. A total of 32 T2-weighted spin-echo images were acquired with the echo spacing equal to 1.303 ms. Eight averages were acquired with the repetition time equal to 1 s, giving a total acquisition time of the data set of ca. 9 min. PD maps were acquired with the use of the same pulse sequence and set of parameters, only the value of the repetition time was increased to 10 s. To acquire T1 maps, a two-dimensional single-slice saturationrecovery sequence was used. The image parameters were the same as for the T2 maps acquisition. A total of 16 spin-echo images with different recovery delays (TR) were acquired with the echo time TE equal to 847 ms. Two averages were acquired per image. The total acquisition time of the complete data set was 57 min. For the D2O/H2O exchange measurements, the two-dimensional single-slice single spin-echo sequence was used with the same image parameters as above. The lenses were taken out of AAH and placed into the 10 mm NMR tube filled with 3 mL of D2O. During the process of replacement of H2O molecules in the lens with D2O molecules, the decrease in the 1H NMR signal intensity was observed. A series of T2-weighted images were acquired during this process. A total of 4 averages were acquired with a repetition time of 1 s. The acquisition time of one image in the series was 4 min 18 s. The number of images acquired depended on the H2O/D2O replacement rate for a particular lens, and the images were acquired continuously while the 1H NMR signal of H2O molecules in the lens persisted. For the 31P MRI diffusion measurements, a two-dimensional single-slice single spin-echo sequence was utilized. The lenses were taken out of AAH and placed into the 10 mm NMR tube filled with phosphate buffer solution (4 M, pH 6.4). During the process of diffusion of 31P-containing species in the lens, the 31P NMR signal intensity was observed to grow. A series of T2-weighted images were acquired during the process. The slice thickness was 2.2 mm and the field of view was ca. 6.4  1 cm2 with the 64  10 image matrix, which corresponds to the in-plane spatial resolution of ca. 1  1 mm2. The echo time was equal to 995 ms. A total of 128 averages were acquired with a repetition time of 0.5 s. The acquisition time of one image was equal to 10 min 47 s. The number of images acquired depended on the diffusion rates for a particular lens, and the images were acquired continuously while any changes in the 31 P NMR signal intensity in the lens were observed. Before reconstruction of the image, a sine filter was applied to reduce the noise level.

the S(t) dependence with an exponential function yields the value of T1. The fitting procedure was repeated for each image pixel, and then the corresponding T1, T2 and PD maps of water in the lens were constructed. In order to compare the experimental results obtained for different strains and ages, the following procedure was used. For every acquired image, a one-dimensional central profile was analyzed. The center of the profile was determined as described below, and its abscissa was set to 0. The distance along the profile was normalized in such a way that the radius of the lens was equal to unity. Then the profiles corresponding to the lenses of the same strain and age were averaged. The center of the profile corresponds to the minimum of a measured value (T1, T2 or PD). In order to determine this minimum, the first derivative was approximated as follows: f0 (xi) ¼ (f(xiþ1)  f(xi1))/(xiþ1  xi1), where (xi, f(xi)) are the experimental data points. Then the calculated f0 (x) was fitted with a straight line. The point where this line intersects the line f0 (x) ¼ 0 was treated as the minimum of the profile. The averaging procedure for N profiles was as follows: all data points from these N profiles were added to a list and sorted in the order of increasing abscissa. Then the averaged profile was constructed using the conventional moving average procedure, with an unweighted mean of N data points calculated at each step. The kinetics of D2OeH2O exchange in the lens immersed in D2O solution was simulated with the use of a simple mathematical model of diffusion of water out of a homogeneous sphere with an effective diffusion coefficient D and initially uniform water content. The concentration of H2O at the external surface of the sphere is always zero. The one-dimensional equation for radial diffusion was numerically solved subject to the appropriate initial and boundary conditions:

  vcðr; tÞ 1 v vcðr; tÞ r2 D ; ¼ 2 vt vr r vr

cðr; 0Þ ¼ 1; 0  r  1

(1)

cð1; tÞ ¼ 0; t  0;

where c is the concentration of H2O molecules, r is the distance from the sphere center, and t is time. An evolution in time of a onedimensional radial concentration profile, c(r), was calculated. To account for the non-uniform initial distribution of H2O within the lens and the relaxation weighting of the profile intensity that changes with r, the calculated profiles were multiplied by an even polynomial function of the form f(r) ¼ A þ Br2 þ Er4. The coefficients in this function were initially adjusted to reproduce the 1 H NMR signal intensity profile extracted from a relaxationweighted image of a similar lens. To take into account the finite image acquisition time, the calculated profiles were time-averaged accordingly. The resulting calculated series of profiles were compared with the experimental data sets. The variable parameters of the fitting procedure were the value of the absolute intensity scaling factor and the effective diffusivity D. The best fit of the calculated set of profiles to the experimental one was searched for using the LevenbergeMarquardt least-squares algorithm.

2.4. Data processing and presentation 3. Results For the water T1 and T2 mapping experiments, the data sets were processed as follows. For every pixel of an image, the signal intensity S as a function of time t was fitted with the general equation S(t) ¼ S0 þ PD exp(TE/T2) (1  exp(TR/T1)). In the T2 mapping experiments, TE is the variable parameter (TE ¼ t) while RD is kept constant. The fit of the S(t) dependence with an exponential function yields the values of T2 and the proton density (PD) if the value of TR is sufficiently long. In the T1 mapping experiments, TR is the variable parameter (TR ¼ t) while TE is kept constant, and the fit of

The acquisition of MRI maps of the rat lenses was performed with the use of two methods: the T1 maps were obtained using the saturation-recovery pulse sequence, while the measurements of proton density (PD) and T2 relaxation times were performed using the CPMG method. Fig. 1a shows the typical saturation-recovery kinetics obtained for the selected pixels in the central and peripheral parts of a 3-month-old Wistar lens immersed in AAH solution (the selected pixels are shown by small rectangles in Fig. 2b).

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Fig. 1. Normalized relaxation curves demonstrating the measurements of T1 (a) and T2 (b) times in selected pixels from the cortex (open circles) and nucleus (solid circles) regions of a 3-month-old Wistar lens. Solid lines show the exponential fits with the time constants: T1(cortex) ¼ 1.56 s, T1(nucleus) ¼ 0.9 s, T2(cortex) ¼ 18 ms, T2(nucleus) ¼ 2.14 ms.

The obtained T1 values for this kinetics are 0.90  0.09 s and 1.56  0.04 s. Fig. 1b demonstrates the decays of the spin echo signals from the same regions. The exponential fit gives the T2 relaxation time values for these curves equal to 2.14  0.09 ms and 18.0  0.3 ms. Due to the optimization of the pulse sequence performed to shorten the echo times TE (see Experimental section), the first point in the graphs corresponds to the echo time of 1.3 ms, and even for very short relaxation times at least four experimental points above the noise level can be detected in the signal decay curve. Fig. 2 shows T1, PD and T2 maps obtained for a 3-month-old Wistar lens immersed in AAH, as well as T1, PD and T2 profiles along the lens diameter. The PD profile is normalized in such a way that the PD values for the AAH solution outside the lens were near unity. It has been shown earlier (Hills et al., 1989; Moffat and Pope, 2002b; Moffat et al., 2002) that the major mechanism of the nuclear spin relaxation of water in protein-containing media is the exchange of

water with NH and OH protons of the amino acid side chains in proteins, and the main factor determining the relaxation time of water protons in biological tissues such as the lens is the protein concentration. This implies that all three profiles in fact reflect the same property of the tissue e namely, the percentage of water. Jones and Pope (2004) reported that in the lens homogenates the rate of longitudinal relaxation 1/T1 linearly depends on the dry-towet mass ratio (DW), while the rate of transverse relaxation 1/T2 shows an exponential dependence:

1=T1 ¼ A1 þ B1  DW

(2)

1=T2 ¼ A2  expðB2  DWÞ

(3)

The water content profiles calculated from the experimental T1 and T2 profiles according to Eqs. (2) and (3) are shown in Fig. 3. The parameters A1 ¼ 0.1 s1, B1 ¼ 1.5 s1, A2 ¼ 8.0 s1 and B2 ¼ 6.0 were

Fig. 2. Upper row: T1 (a), PD (b) and T2 (c) maps of a 3-month-old Wistar lens immersed in AAH. Bottom row, black lines: T1 (a), PD (b) and T2 (c) profiles of the same lens; red line: T2 profile of a 3-month-old Wistar lens in air. Small white rectangles in PD map show the pixels used for the demonstration of typical relaxation curves shown in Fig. 1. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Water content

0,8 0,6 0,4 0,2 0,0

-2

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X (mm) Fig. 3. Water content profiles for a 3-month-old Wistar lens immersed in AAH obtained from MRI data. Black squares e PD values inside the lens relatively to the PD value outside the lens; blue triangles e water content calculated from T1 profile according to Eq. (2) with A1 ¼ 0.1 s1 and B1 ¼ 1.5 s1; red circles e water content calculated from T2 profile according to Eq. (3) with A2 ¼ 8.0 s1 and B2 ¼ 6.0. Horizontal black lines show the average water content in cortex and nucleus obtained by the direct measurement of wet and dry weights. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

fitted to match the PD profile. An excellent agreement between the three profiles shown in Fig. 3 confirms that indeed, the water percentage in a lens is the main factor determining T1 and T2 relaxation times, and both T1 and T2 maps reflect the water content distribution inside the lens. At the same time, T2 measurements have a significant advantage over PD and T1 measurements: for the acquisition of correct PD and T1 data, the repetition time of a pulse sequence should be sufficiently long to allow the spin system to return to equilibrium before the next signal acquisition is started. If this condition is not met, the regions with longer T1 relaxation times will give weaker signals due to the partial signal saturation. The T2 measurements do not have such limitation, and allow for a much faster data acquisition. For example, the total acquisition times are 57 min for a T1 map (Figure 2a), 1 h 26 min for a PD map (Fig. 2b), and only 9 min for a T2 map (Fig. 2c). The control experiments demonstrated that making repetition time longer than 1 s does not influence the T2 profiles, while shortening it below 10 s does distort the PD profiles. Besides, since the 1/T2 values depend

on the water percentage exponentially, they are more sensitive to the changes in water content. Indeed, the PD and T1 values in the lens cortex and nucleus differ by approximately a factor of two, while the difference in T2 values exceeds an order of magnitude. Therefore, all further measurements of the water content distribution in this work were performed with the use of T2 measurements only. In a control experiment, we performed direct measurements of the water content in 3-month-old Wistar lenses. For this purpose, the nuclei of three 3-month-old Wistar lenses were separated from the cortexes, the wet and the dry weights of each portion were measured (see Experimental part), and the water contents in the nucleus and the cortex of each lens were determined. The averaged values are 0.34  0.01 and 0.62  0.04, respectively. These values, shown in Fig. 3 with horizontal lines, are in a very good agreement with the water content profiles obtained by MRI methods, and with the previously reported data (Kopylova et al., 2011) that the total protein content of the 3-month-old Wistar lens amounts to approximately 50% of the lens wet weight. We have also checked if the presence of AAH solution in the sample tube is necessary to correctly measure the T2 values within the lens. Fig. 2c shows an additional T2 profile obtained with the lens placed in a dry sample tube. To prevent the lens drying, a small piece of wet cotton wool was placed at the top of the tube. The comparison of profiles in Fig. 2c shows that no significant sample drying occurs during the signal acquisition, and the T2 maps of the lens in water and in air practically coincide. No significant difference was also revealed between the T2 maps obtained at ambient temperature and at 35  C. Therefore, the T2 measurements were performed at ambient temperature (in our case, typically 25e28  C) in dry sample tubes immediately after removing the lens from AAH solution. Fig. 4 demonstrates the T2-maps of three Wistar and three OXYS lenses. The rat lenses have an almost spherical shape, and the lens size depends on both age and strain: with age, the lens diameter significantly increases; at the same time, the OXYS lenses are slightly smaller than the Wistar ones of the same age. The latter observation should be attributed to the phenotypic difference between the two strains: the OXYS rats are in general somewhat smaller than their predecessor Wistar rats (Marsili et al., 2004). Similar T2-maps were obtained for 4e8 lenses for each age and strain.

Fig. 4. T2 maps of Wistar and OXYS lenses of different age.

An attempt to acquire an apparent diffusion coefficient (ADC) map for the lens of a 6 months old Wistar rat was made. The PGSTE NMR sequence (Callaghan, 1991; Price, 1998) was utilized to acquire a series of diffusion-weighted images. Unfortunately, low diffusion rates and high nuclear spin relaxation rates in the lens made our measurements rather inaccurate. The signal attenuation owing to diffusion weighting was found to be only about 2% because of the low diffusion rates, and extremely high relaxation rates caused signal attenuation that reduced the signal-to-noise ratio. These factors did not allow us to acquire reliable quantitative information about the ADC in any acceptable time. The only available estimation obtained from these measurements is that the diffusion coefficient in the central part of the lens is not higher than 3  106 cm2/s, i.e. at least one order of magnitude lower than that for free water. Real-time measurements of the water transport diffusion in the rat lens were performed by immersing a lens in deuterated water and monitoring the evolution of relaxation-weighted images of the lens. D2OeH2O exchange causes decrease of the proton density and the attenuation of the NMR signal, so the kinetics obtained reflects the D2O penetration into the lens. Although the samples were placed inside the NMR probe immediately after lens immersion in D2O and the first image has been acquired within 5e10 min thereafter, the kinetics of D2O penetration in 20-day-old lenses was too fast for reliable measurements. Therefore, the experiments were performed for 3-month-old and 12-month-old lenses only. Fig. 5 shows images of a 3-month-old Wistar lens 10, 23, 40 and 75 min after immersion in D2O. The kinetics of D2OeH2O substitution in the two regions of interest, marked in Fig. 5 by rectangles and corresponding to the lens cortex and nucleus, is given in Fig. 6. The exponential fit of these data gives rate constants of (4.2  0.1)  104 s1 and (2.5  0.1)  104 s1 for the cortex and nucleus regions, respectively. Simulation of D2OeH2O exchange in the lens was performed with the use of a simple model of diffusion of water out of a homogeneous sphere as described in the Experimental section. Apparently, this model is oversimplified since the water diffusion inside the lens is highly anisotropic and inhomogeneous (Moffat and Pope, 2002a), and cannot be used for a correct description of water transport. However, the advantage of the simple model is that it has only two variable parameters, the diffusion coefficient and the scaling factor. This allows for a simple semi-quantitative comparison of the water diffusion rates in lenses of different

Fig. 5. MRI images of 3-month-old Wistar lens 10, 23, 40 and 75 min after the immersion in D2O. Small white rectangles in the upper left image show the pixels used for the demonstration of typical kinetic curves in Fig. 6.

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Time (min) Fig. 6. Normalized kinetics of D2OeH2O substitution in the cortex (open circles) and nucleus (solid circles) of 3-month-old Wistar lens immersed in D2O. Solid lines show exponential fits with the rate constants 4.2  104 s1 and 2.5  104 s1 for the cortex and nucleus regions, respectively.

strains and ages. The measurements were performed for Wistar and OXYS 3-month-old and 12-month-old lenses, two lenses of each type (8 lenses total). The relaxation-weighted proton signal profiles were obtained with the time spacing of 4 min 18 s. A typical set of experimental and calculated profiles is shown at Fig. 7 (only every fourth profile is shown), and the values of the calculated diffusion coefficients are given in Table 1. It is important to note that the kinetics of D2OeH2O exchange was measured in non-physiological conditions: the temperature (approximately 27  C) was lower than the body temperature, and the D2O solution in which lenses were immersed did not contain salts present in the aqueous humor. Some previous publications (Mathias and Rae, 1985, 2004; Mathias et al., 2007; Vaghefi et al., 2011, 2012) indicate that microcirculation driven by ion currents may play an important role in the water transport through the lens, though the contribution of this mechanism remains unclear. If, indeed, the contribution of the active diffusion is high, the water transport under physiological conditions should be significantly faster. To check this hypothesis, we performed control experiments, in which one lens of 2-month-old rat was immersed in D2O, and the other lens, belonging to the same rat, into D2O-based AAH solution. Then the kinetics of D2OeH2O exchange was measured at T ¼ 35  C, and the diffusion coefficient values were obtained by fitting of Eq. (1) to the experimentally detected evolutions of the proton signal profiles. The same procedure has been repeated for three pairs of lenses. The average values of the obtained diffusion coefficients are D(D2O) ¼ (2.4  0.3)  106 cm2/s and D(AAH-D2O) ¼ (2.3  0.4)  106 cm2/s; typical kinetics curves obtained for the lens central pixels are shown in Fig. 8. The kinetic curves and the obtained diffusion coefficient values demonstrate that the rates of the D2O penetration into lenses immersed in D2O (non-physiological conditions) and D2O-based AAH (physiological conditions) are very similar. That testifies that the passive diffusion is the major mechanism of the water transport in lenses under study. More detailed report on the influence of experimental conditions on the rate of water transport in a lens will be published in a separate paper. We have also made an attempt to measure the rate of the phosphate diffusion in the lens using 31P NMR imaging. For this purpose, a 3-month-old Wistar lens was immersed in 4 M phosphate buffer (pH 6.4), and the kinetics of phosphate penetration inside the lens was monitored. Apparently, the acquisition of 31 P NMR images is significantly more challenging than the 1H

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Ti m e

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Fig. 7. Experimental (left) and simulated (right) temporal evolution of the relaxation-weighted proton signal profiles for a 3-month-old Wistar lens immersed in D2O. The time interval between profiles is 17.2 min.

Table 1 Diffusion coefficients calculated from the simulation of the D2OeH2O exchange in the lenses of 4 Wistar and 4 OXYS rats. Rat strain

Diffusion coefficient D, cm2/s 3 Months

Wistar OXYS

(1.3 (1.2 (1.4 (1.5

   

0.1) 0.1) 0.2) 0.1)

After the immersion, the rate of the D2OeH2O exchange inside the lens in the presence of phosphate was measured. It was found that the rate of D2OeH2O exchange in the presence of phosphate is only slightly slower than that in pure D2O. For example, the monoexponential rate constant of the H2O signal decay in the lens center in the presence of phosphate was (1.61  0.06)  104 s1, i.e. approximately 1.4-fold lower than that without phosphate (see above). Thus, high concentrations of phosphate did not give a significant effect on the rate of water transport through the lens, and the much slower diffusion of phosphate in comparison with water should be attributed exclusively to the properties of phosphate e either the relatively large size of phosphate ions or their interactions with the medium. 4. Discussion Since the lenses used in the present study are of different sizes, to compare them it is convenient to present the experimental data with the lens dimensions expressed in units of the lens radius. The recalculation was performed for T2 profiles of Wistar and OXYS lenses of different ages, the profiles obtained for the same age and strain were averaged (4e8 lenses for each age and strain). The results are shown in Fig. 10. Since the shape of lenses may slightly differ from a spherical one, the data scattering at the very edges of the profiles was much higher than in all other parts of a lens. The voxels at the lens edges could include space from outside the lens, which would also result in the increased data scattering at the profile edges. For this reason, the profiles in Fig. 10 are shown in the

Signal intensity

measurements due to the sensitivity problem: the spin density of 31 P even in concentrated aqueous solutions is 25-fold lower than that of protons, and the magnetogyric ratio for 31P is 2.5-fold lower. Besides, the diameter of the receiving coil in our 31P NMR probe is a factor 1.7 larger than the proton probe, which makes the filling factor at least 3-fold lower. All factors combined, the sensitivity of 31 P MRI measurements is two to three orders of magnitude poorer than that of 1H measurements. To achieve an acceptable signal-tonoise level, we had to use significantly larger sizes of the localized volume elements (1  1  2.2 mm) and much longer experiment times (each echo signal was accumulated 128 times). Fig. 9 shows the kinetic traces of 31P spin density increase in two neighboring localized volumes, attributed to the cortex and nucleus regions. The data in Fig. 9 are given relative to the 31P NMR signal intensity of solution outside the lens, where it was taken to be equal to unity. We should note that under our experimental conditions, the size of the volume elements used is comparable to the lens size (a sphere of 4 mm diameter), and the terms “cortex” and “nucleus” have only relative meaning: for example, the “cortex” volume element includes part of the nucleus region as well as free solution from the sample outside the lens. Thus, the 31P data should be considered only qualitatively. Fig. 9 demonstrates that the diffusion of phosphate inside the lens is much slower than that of water. The increase of 31P signal in the “cortex” can be fitted by an exponential with the rate constant of (3.7  0.2)  105 s1, and in the “nucleus” e of (2.5  0.2)  105 s1. This is an order of magnitude slower than the rates observed in the D2OeH2O exchange measurements. The size of phosphate ions (MW ¼ 95  97 D) is significantly larger than that of water molecules, so their movement through the cell membranes should be slower. An alternative explanation of the slow phosphate diffusion observed in our experiments might be the influence of the high salt concentration on the diffusion rate. In the recent work of Vaghefi et al. (2011), the deceleration of the water diffusion through a lens in the presence of high concentrations of Kþ ions was observed. The authors attributed this observation to the disruption of the lens ionic homeostasis which might be playing an essential role in the active water influx in the lens. To check this possibility, we performed a control experiment in which a rat lens was immersed in a D2O solution containing 4 M phosphate buffer.

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106 106 106 106

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Fig. 8. Normalized kinetics of D2OeH2O substitution in the center of 2-month-old Wistar lens immersed in D2O (squares) and in D2O-based AAH (circles). Solid lines show the calculated kinetic curves with D ¼ 2.12  106 cm2/s (D2O) and D ¼ 2.06  106 cm2/s (D2O-based AAH); the calculations were performed according to Eq. (1) to fit the whole set of data of temporal evolution of proton signal profiles.

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Time (hours) Fig. 9. Kinetics of phosphate penetration inside the 3-month-old Wistar lens immersed in 4 M phosphate buffer obtained for the cortex (open circles) and nucleus (solid circles) regions. The signal intensity is given relatively to the 31P signal outside the lens. Solid lines show exponential fits with the rate constants 3.7  105 s1 and 2.5  105 s1 for the cortex and nucleus regions, respectively.

range from 0.95r to 0.95r, where r is the lens radius. The profiles obtained for 3-month-old and 18-month-old lenses are characterized by a relatively flat T2 distribution in the central part of the lens (approximately 70% of the lens diameter) and a steep increase in the T2 values with increasing radial coordinate within the cortex. In the youngest (20-day-old) lenses these features are less pronounced. Partly, this observation should be attributed to insufficient spatial resolution of the method employed: although the inplane image resolution was sufficiently high (78  154.5 mm2 pixels), the slice thickness was 1 mm, and for small spherical objects such as 20-day-old lenses (the diameter is about 3 mm) the slice could partially include different parts of the lens; however, this effect is minor, it results in only a small flattening of the T2 profiles. The following features of the profiles should be noted: 1) there is an obvious T2 gradient along the lens diameter with T2 decrease toward the center of the lens; 2) this gradient becomes more pronounced with age: the T2 values at the profile edges (outer cortex) only slightly depend on age, while in the inner cortex the decrease in T2 values becomes steeper with age, and in the lens nucleus the nuclear relaxation is faster in old lenses; 3) no significant difference has been found between T2 profiles of Wistar and OXYS lenses of the same age. In some previous T2 measurements on the whole lenses, a multiexponential transverse relaxation has been observed (Stankeiwicz

et al., 1989; Lerman and Moran, 1989; Rácz et al., 2000). The components of the signal decay were attributed to the different states of water, with the pool of “free” water corresponding to the slow component, and the faster components corresponding to water bound to the lens proteins. This interpretation was questioned by Moffat and Pope (2002b) who have shown that the multiexponential spin-echo decay should be rather attributed to the signals from different parts of the lens with different water contents, with higher water contents corresponding to the longer-lived components in the signal decay measured without spatial resolution. Our results confirm this explanation: Fig. 3 clearly demonstrates that the proton relaxation times T1 and T2 are mostly determined by the water content in each particular voxel. The T2 values in the nucleus of the rat lens reported here (about 2 ms) are significantly lower than those in the nuclei of human and porcine lenses (15e25 m) (Moffat and Pope, 2002b; Moffat et al., 2002; Patz et al., 2007). This means that the crystallins in the rat lenses are packed more tightly, and the water percentage is lower. Indeed, the microradiography measurements demonstrate (Fagerholm et al., 1981) that the dry mass content of the human lens varies from 0.2 g/cm3 in the outer cortex to 0.45 g/cm3 in the nucleus, i.e. the water content exceeds 50% even in the lens core. The water content of the bovine lens was reported to be approximately 80% in the outer cortex and 60% in the nucleus (Babizhayev et al., 2002). In the rat lenses under study, the water content in the lens center is as low as 30% (Fig. 3). The water content along the optical axis of the rat lens varies very significantly, so one can conclude that the refractive index gradient (GRIN) in the rat lens is steeper than that in the human lens. Despite the lower water content in the rat lens as compared to the human lens, the diffusion of water through the lens seems to proceed with the similar rate: the kinetic curves of the D2OeH2O exchange obtained in the present work are similar to those reported for the human lens (Moffat et al., 1999). This confirms that the “bottleneck” for diffusion through the lens is the water penetration through the cell membranes rather than the intracellular water movement. Our results also confirm the finding of Moffat et al. (1999) that the diffusion rate decreases with age: the average diffusion coefficient estimated in the present work for 3month-old lenses is approximately 1.6-fold greater than that for 12-month-old lenses. The attempt to measure the phosphate diffusion through the lens by MRI was made for the first time. Due to the sensitivity problem, the obtained results can bear only qualitative significance; nevertheless, the comparison of proton and phosphorus kinetic measurements (Figs. 6 and 9) unambiguously testifies that

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Fig. 10. Average T2 profiles of Wistar (left) and OXYS (right) lenses. Black lines e 20-day-old, red lines e 3-month-old, blue line e 18-month-old lenses. Error bars show typical standard errors. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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the phosphate diffusion into the lens is significantly slower than that of water. It is interesting to note that in the 31P kinetic experiments, the level of 31P MRI signal inside the lens reached only 20e30% of that outside the lens even after 60 h of incubation. We suppose that this observation should be attributed to the low water content inside the lens. Even under equilibrium in phosphate concentrations inside and outside the lens, the amount of phosphate per unit volume is expected to be proportional to the water content, and inside the lens it is lower than outside. We have not found any significant difference between the T2 profiles of OXYS and Wistar lenses (Fig. 10), even though OXYS rats are significantly more prone to the cataract development. Some previous data (Rácz et al., 2000; Lizak et al., 2000) indicate that the water content and state in the cataractous lens differ from that in the normal lens, which results in the changes of the transverse relaxation time. Our results do not confirm this hypothesis, at least with respect to the rat lenses. No significant difference between OXYS and Wistar lenses was observed in the water diffusion rates either. Actually, the diffusion coefficients for the water transport through the OXYS lenses is even somewhat higher than that for Wistar lens, although the difference is only slightly above the experimental error. The impediment to water diffusion through the lens is often considered to be one of the main causes of cataractogenesis (Sweeney and Truscott, 1998; Moffat et al., 1999; Truscott, 2005): slower diffusion causes a decrease of antioxidant level in the lens core and the increase of oxidized species concentration. This results in oxidative stress development, extensive protein modification, and lens opacification. The characteristic feature of OXYS rats studied in the present work is the excessive generation of reactive oxygen species (Marsili et al., 2004), which is responsible for the accelerated aging of the animals in general, and for the early cataract onset in particular. Thus, the obtained results imply that slow water diffusion through the lens can facilitate the oxidative stress conditions and cataract development, but not the other way around. Quite likely, the enhanced generation of reactive oxygen species in OXYS rats either does not influence the water flux through the lens, or even accelerates it due to a compensatory response. 5. Conclusions The results obtained in the present work demonstrate that MRI methods can be successfully applied to the study of fine structure and physical properties of such small biological objects as rat lenses. A good match between PD, T1 and T2 maps proves that all three reflect the same property of the lens tissue e namely, the distribution of the water content inside the lens. The water content in the core of the rat lens is only 30%, which is much lower than that in human, porcine and bovine lenses. A significant difference between water contents in the cortex and nucleus results in a strong refractive index gradient, providing a high optical power of the rat lens. The following age-related changes in the rat lens have been revealed: A) the water content in the lens nucleus decreases with age, and the water content gradient in the lens cortex becomes steeper. These changes are more pronounced during the lens maturation period between the ages of 3 weeks and 3 months. B) The rate of water diffusion through the lens decreases with age. Our estimate is that the average diffusion coefficient in a 3-month-old lens is approximately 1.5 times higher than that in a 12-month-old lens. The observed age-related changes are in a good correlation with similar changes reported earlier for the human lens (Moffat et al., 1999, 2002; Moffat and Pope, 2002a; Jones et al., 2005; Kasthurirangan et al., 2008). At the same time, no difference in the water content distribution has been found between Wistar and

OXYS rat lenses of matching ages, while the cataract onset in the OXYS lenses occurs much earlier due to enhanced generation of reactive oxygen species. This finding implies that the cataract development does not lead to significant changes in the water content distribution inside the lens. The water diffusion in the OXYS lens is slightly faster than in the Wistar lens, which might probably be attributed to a compensatory response to the oxidative stress. Acknowledgments We thank Prof. N.G. Kolosova and Dr. N.A. Stefanova for providing the experimental animals. This work was supported by RFBR (Projects 11-04-00143, 11-03-00296 and 12-04-31244), the Division of Chemistry of RAS, the Siberian Branch of RAS (integration projects 57 and 60), the Government of the Russian Federation (grants 11.G34.31.0045 and 8094), and the President of the Russian Federation (grant NSh-2429.2012.3). References Andley, U.P., 2007. Crystallins in the eye: function and pathology. Prog. Retin. Eye Res. 26, 78e98. Babizhayev, M.A., Nikolayev, G.N., Goryachev, S.N., Bours, J., 2002. NMR spin-echo studies of hydration properties of the molecular chaperone a-crystallin in the bovine lens. Biochim. Biophys. Acta 1598, 46e54. Beebe, D.C., Truscott, R.J.W., 2010. Counterpoint: the lens fluid circulation model e a critical appraisal. Invest. Ophthalmol. Vis. Sci. 51, 2306e2310. Bloemendal, H., de Jong, W., Jaenicke, R., Lubsen, N.H., Slingsby, C., Tardieu, A., 2004. Ageing and vision: structure, stability and function of lens crystallins. Prog. Biophys. Mol. Biol. 86, 407e485. Callaghan, P., 1991. Principles of Nuclear Magnetic Resonance Microscopy. Oxford University Press Inc., New York. Donaldson, P.J., Musil, L.S., Mathias, R.T., 2010. Point: a critical appraisal of the lens fluid circulation model e an experimental paradigm for understanding the maintenance of lens transparency? Invest. Ophthalmol. Vis. Sci. 51, 2303e2306. Fagerholm, P.R., Philipson, B.T., Lindström, B., 1981. Normal human lens e the distribution of proteins. Exp. Eye Res. 33, 615e620. FitzGerald, P.G., Goodenough, D.A., 1986. Rat lens cultures: MIP expression and domains of intercellular coupling. Invest. Ophthalmol. Vis. Sci. 27, 755e771. Fletcher, A.E., 2010. Free radicals, antioxidants and eye diseases: evidence from epidemiological studies on cataract and age-related macular degeneration. Ophthalmic Res. 44, 191e198. Foster, A., Gilbert, C., Johnson, G., 2008. Changing patterns in global blindness: 1998e2008. Community Eye Health 21, 37e39. Garner, L.F., Smith, G., Yao, S., Augusteyn, R.C., 2001. Gradient refractive index of the crystalline lens of the Black Oreo Dory (Allocyttus Niger): comparison of magnetic resonance imaging (MRI) and laser ray-trace methods. Vis. Res. 41, 973e 979. Giblin, F.J., 2000. Glutathione: a vital lens antioxidant. J. Ocul. Pharmacol. Ther. 16, 121e135. Goodenough, D.A., Dick II, J.S.B., Lyons, J.E., 1980. Lens metabolic cooperation: a study of mouse lens transport and permeability visualized with freezesubstitution autoradiography and electron microscopy. J. Cell Biol. 86, 576e589. Goodenough, D.A., 1992. The crystalline lens. A system networked by gap junctional intercellular communication. Semin. Cell Biol. 3, 49e58. Graw, J., 1997. The crystallins: genes, proteins and diseases. Biol. Chem. 378, 1331e 1348. Hills, B.P., Takacs, S.F., Belton, P.S., 1989. The effects of proteins on the proton N.M.R. transverse relaxation times of water. I. Native bovine serum albumin. Mol. Phys. 67, 903e918. Jones, C.E., Pope, J.M., 2004. Measuring optical properties of an eye lens using magnetic resonance imaging. Magn. Reson. Imaging 22, 211e220. Jones, C.E., Atchison, D.A., Meder, R., Pope, J.M., 2005. Refractive index distribution and optical properties of the isolated human lens measured using magnetic resonance imaging (MRI). Vis. Res. 45, 2352e2366. Jones, C.E., Atchison, D.A., Pope, J.M., 2007. Changes in lens dimensions and refractive index with age and accommodation. Optom. Vis. Sci. 84, 990e995. Kasthurirangan, S., Markwell, E.L., Atchison, D.A., Pope, J.M., 2008. In vivo study of changes in refractive index distribution in the human crystalline lens with age and accommodation. Invest. Ophthalmol. Vis. Sci. 46, 2531e2540. Kasthurirangan, S., Markwell, E.L., Atchison, D.A., Pope, J.M., 2011. MRI study of the changes in crystalline lens shape with accommodation and aging in humans. J. Vis. 11, 1e16. Kolosova, N.G., Lebedev, P.A., Aidagulova, S.V., Morozkova, T.S., 2003a. OXYS rats as a model of senile cataract. Bull. Exp. Biol. Med. 136, 415e419. Kolosova, N.G., Lebedev, P.A., Fursova, A.Zh., Morozkova, T.S., Gusarevich, O.G., 2003b. Prematurely aging OXYS rats as an animal model of senile cataract in human. Adv. Gerontol. 12, 143e148.

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