Ординатура / Офтальмология / Английские материалы / Progress in Lens and Cataract Research_Hockwin_2002

Fig. 1. Culture conditions of the lens obtained from chick embryos.

chick embryos treated with glucocorticoids [2], were incubated with steroids. The results indicate that androgen, estrogen and mineralocorticoid as well as glucocorticoids can cause a loss of transparency of the lens in a different way from that in the in ovo system (whole body system).

Material and Methods

Chemicals

Dexamethasone, prednisolone, hydrocortisone, hydrocortisone succinate sodium (HC), cortisone, testosterone, methyltestosterone, estradiol, ethinylestradiol, 19-nor-ethisterone and aldosterone were obtained from Sigma Chemical Co. (St. Louis, Mo.). All other reagents were of analytical grade.

Animals

Chick embryos were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Fertile white leghorn eggs were used for all experiments. One-day-old fertile white leghorn eggs were purchased from a local hatchery and incubated in a humidified incubator at 37.5 °C.

Lens Culture System

Lenses were carefully excised from 16-day-old chick embryos. Each lens was placed on a nylon mesh, fixed in the middle of 24-well multidishes (Falcon No. 3047, Becton Dickinson, Lincoln Park, N.J.), and was covered with medium slightly over the posterior part of the lens (fig. 1). Culture medium consisted of M 199 with Earle’s salt (Sigma Chemical Co.), 15% chicken serum (Flow Laboratories, McLean, Va.), 25 mM HEPES, 83 mM glucose, 1 mM ascorbic acid and antibiotics (100 U/ml penicillin and 0.1 mg/ml streptomycin). A stock solution (0.1 M in ethanol) of each steroid hormone shown in figure 2 was added to culture medium to the desired concentrations with a final ethanol concentration of 0.1%. After the opaque lenses were removed for 24 h preculture, clear lenses were cultured for an additional 48 h at 37 °C with various concentrations of steroid hormones or with ethanol alone (0.1%) as a control in a humidified atmosphere containing 5% CO2. After the culture, the lenses were visually classified as described previously except stage IV and V were not separated [5–7].

Fig. 2. Chemical structure of typical steroid hormones.

Developing Chick Embryo System

Glucocorticoids and other steroid hormones were administered to 15-day-old developing chick embryos as described previously [5–7]. Briefly, HC dissolved in water and other steroids dissolved in 5% acetone in water were administered to the embryos (0.25 mol/200 l). Lenses were removed from chick embryos at 48 h after the steroid administration, and visually classified as described previously except stage IV and V were not separated [5–7].

Results

When 0.25 mol of HC were administered to 15-day-old developing chick embryos, most of their lenses became opaque within 48 h after the steroid treatment (fig. 3a). In the lens culture system, when lenses were incubated with 1 10 4 M dexamethasone for 48 h, the nuclear region in these lenses became opaque similar to the developing chick embryo system (fig. 3b). Then, the lenses were incubated with several steroids for 48 h and examined. For the experiments, the selected steroids were as follows: testosterone as androgen, estradiol as estrogen, aldosterone as mineralocorticoid, dexamethasone and hydrocortisone as glucocorticoids. Cortisone was also examined. As shown in table 1, all steroids tested caused the loss of transparency of lenses at similar concentration over 3 10 5 M, unrelated to their biological activities.

Fig. 3. Lenses treated with glucocorticoid in the whole body system (a) and in the culture system (b). a Fifteen-day-old chick embryos were given 0.25 mol of HC and their lenses were removed from chick embryos at 48 h after HC treatment. b Lens was obtained from 16-day-old chick embryos and cultured for 48 h with 1 10 4 M dexamethasone (for details see Material and Methods).

Table 1. Effect of steroids on cultured lens of developing chick embryos

Data indicate the number of lenses with percentages in parentheses.

When 0.25 mol of HC and prednisolone, and 0.01 mol of dexamethasone were administered to 15-day-old chick embryos, their lenses became cataractous. However, the steroids possessing no glucocorticoid activity such

Table 2. Incidence of cataractous lenses in steroid-treated developing chick embryos

Data indicate the number of embryos with percentages in parentheses.

as cortisone, methyltestosterone, estradiol, ethinylestradiol, progesterone and 19-nor-ethisterone did not cause cataract (table 2).

Discussion

We have demonstrated by using developing chick embryos that only biological active glucocorticoids, C-21 steroids with hydroxyl residue on C-11 position, can cause cataract [3, 5, 6]. Although glucocorticoids showed cataractogenic activity depending on biological potency as described previously, androgen, estrogen and progestin did not cause cataract. However, other than the whole body system, when the isolated chick lenses were cultured in the dishes, their lenses could become opaque in the presence of testosterone, estradiol and aldosterone as well as dexamethasone and hydrocortisone. Cortisone, which is hardly reduced to hydrocortisone [14], did not induce cataract formation in developing chick embryo [6], but caused the loss of lens transparency in the culture system (table 1). Additionally, these steroids including dexamethasone required almost the same concentration, more than 3 10 5 M, to produce the loss of transparency in the lenses. In 15-day-old developing chick embryo,

0.01 mol/egg of dexamethasone, which is approximately 1 10 7 M in the egg (weight approximately 50 g), can produce cataract within 48 h. Therefore, these results demonstrate that the loss of transparency of cultured lens can be induced independently from biological activities of glucocorticoids.

Glucocorticoids cause various metabolic changes in many tissues with glucocorticoid receptor directly and without the receptor indirectly by changed blood components [7, 15, 16]. Therefore, it is quite likely that some adverse effects in tissues after glucocorticoid treatment can be caused by both receptordependent and receptor-independent events. Concerning the cataract formation in animals including humans, there are roughly three mechanisms: the loss of transparency in the lens can be caused by actions of glucocorticoids in lens, by derived factors which are synthesized in other organs than the lens, and by the combined events. It is known that the decreased GSH and over produced LPO in the liver, the high level of LPO in the blood, and the decreased GSH and increased LPO in the cataractous lens occurred in glucocorticoid-induced cataract of developing chick embryos [3–7]. These biochemical events caused by glucocorticoids cannot be observed as a result of the treatment with ascorbic acid [3], insulin [7] and thyroxine [15] which prevent the cataract formation. Among these phenomena, the decreased GSH in glucocorticoid-induced cataractous lenses is well known in various types of cataracts, suggesting the involvement of oxidative stress in the cataract formation [17–19]. Our recent study also showed that the detection of functional glucocorticoid receptor has been unsuccessful in the lens of chick embryos (data not shown). Therefore, LPO may be a risk factor synthesized probably in the liver after glucocorticoid treatment; thus the formation cannot be caused by direct effects of glucocorticoids.

As far as we have managed to establish, there are no papers on whether estrogen, androgen and progestins produce cataract clinically. Glucocorticoids have various physiological and pharmacological activities in the living system. We suppose that steroid-induced cataracts in humans represent one of the adverse effects caused by synergic biological activities of glucocorticoids.

References

1Black RL, Oglasby R, von Sallman L, Bunin J: Posterior subcapsular cataract induced corticoids in patients with rheumatoid arthritis. JAMA 1960;174:166–171.

2Nishigori H, Lee JW, Iwatsuru M: An animal model for cataract research: Cataract formation in developing chick embryo. Exp Eye Res 1983;36:617–622.

3Nishigori H, Hayashi R, Lee JW, Maruyama K, Iwatsuru M: Preventive effect of ascorbic acid against glucocorticoid-induced cataract formation of developing chick embryos. Exp Eye Res 1985;40:445–451.

4Nishigori H, Hayashi R, Lee JW, Yamauchi Y, Iwatsuru M: The alteration of lipid peroxide in glucocorticoid-induced cataract of developing chick embryos and the effect of ascorbic acid. Curr Eye Res 1986;5:37–40.

5Nishigori H, Lee JW, Yamauchi Y, Maruyama K, Iwatsuru M: Analysis of glucose levels during glucocorticoid-induced cataract formation in chick embryos. Invest Ophthalmol Vis Sci 1987; 28:168–174.

6Lee JW, Iwatsuru M, Nishigori H: Glucocorticoid-induced cataract of developing chick embryo as a screening model for anticataract agents. J Ocul Pharmacol Ther 1995;11:533–541.

7Watanabe H, Kosano H, Nishigori H: Steroid-induced short term diabetes in chick embryo: Reversible effects of insulin on metabolic changes and cataract formation. Invest Ophthalmol Vis Sci 2000;41:1846–1852.

8Shui YB, Kojima M, Sasaki K: A new steroid-induced cataract model in the rat: Longterm prednisolone application with a minimum of X-ray irradiation. Ophthalmic Res 1996; 28(suppl 2):92–101.

9Shui YB, Vrensen GF, Kojima M: Experimentally induced steroid cataract in the rat: A scanning electron microscopy study. Surv Ophthalmol 1997;42(suppl 1):s127–s132.

10Manabe S, Bucala R, Cerami A: Nonenzymatic addition of glucocorticoids to lens proteins in steroid-induced cataracts. J Clin Invest 1984;74:1803–1810.

11Bucala R, Gallati M, Manabe S, Cotlier E, Cerami A: Glucocorticoid-lens protein adducts in experimentally induced steroid cataracts. Exp Eye Res 1985;40:853–863.

12Cenedella RJ, Sexton PS, Zhu XL: Lens epithelial contain a high-affinity, membrane steroid hormone-binding protein. Invest Ophthalmol Vis Sci 1999;40:1452–1459.

13Dickerson JE Jr, Dotzel E, Clark AF: Steroid-induced cataract: New perspectives from in vitro and lens culture studies. Exp Eye Res 1997;65:507–516.

14Muscona AA, Piddington R: Enzyme induction by corticoids in embryonic cells: Steroid structure and inductive effect. Science 1967;158:496–497.

15Mayes A: Bioenergetics and the metabolism of carbohydrates and lipids; in Murray RK, Granner DK, Mayes A, Rodwell W (eds): Harper’s Biochemistry, ed 24. Connecticut, Appleton & Lange, 1996, pp 109–284.

16Kosano H, Watanabe H, Nishigori H: Suppressive effects of thyroxine on glucocorticoid (GC)- induced metabolic changes and cataract formation on developing chick embryos. Exp Eye Res, in press.

17Harding J: Cataract: Biochemistry, Epidemiology and Pharmacology. London, Chapman & Hall, 1991.

18Reddy VN: Glutathione and its function in the lens – An overview. Exp Eye Res 1990;50:771–778.

19Spector A: Oxidative stress-induced cataract: Mechanism of action. FASEB J 1995;9:1173–1182.

Hideo Nishigori, Faculty of Pharmaceutical Sciences, Teikyo University, 1091-1, Suwarashi, Sagami-ko, Tsukui-gun, Kanagawa 199-0195 (Japan)

Tel. 81 426 85 3761, Fax 81 426 85 2525, E-Mail h-nishig@pharm.teikyo-u.ac.jp

Hockwin O, Kojima M, Takahashi N, Sliney DH (eds): Progress in Lens and Cataract Research. Dev Ophthalmol. Basel, Karger, 2002, vol 35, pp 169–175

Water Diffusion in the Rabbit

Lens in vivo

Hong-Ming Cheng

Schepens Retina Associates, Boston, Mass., USA

Abstract

Purpose: To examine water diffusion in the crystalline lens and sugar cataracts in the rabbits in vivo.

Materials and Methods: Water self-diffusion in the lens cortices of alloxan-diabetic and galactosemic rabbits was examined with magnetic resonance imaging (MRI). The animals were positioned in a 4.7-tesla animal system in conjunction with a 1-inch surface coil for the eye. Diffusion-weighted MRI was conducted using a pulsed-gradient spin-echo sequence with a gradient strength of 0–6 Gs/cm in the primary and secondary coordinates. Other MRI parameters included TR (repetition time)/TE (echo time) 2,000/10 ms, a field of view of 4 cm, and a 256 128 matrix.

Results: There appeared an increase in water relaxation resulting in an increase of % (equatorial cortex depth)/(lens long axis) from 18 in the lenses of normal rabbits to 30.4 and 39.9 in the lenses of galactosemic and diabetic rabbits, respectively. In addition, water diffusion changed in the lens of the diabetic rabbit with an increasing intracellular fluidity along the long axis of the cortical fibers, for example, the diffusion coefficient changed from a normal of 0.48 to 0.96 10 5 cm2 s 1 in the lens of the diabetic rabbit. These results showed altered water mobility due to subcellular disturbances occurring before any apparent lens opacities. Further, there also was an increase in the water diffusivity in the aqueous humor from a normal of 1.77 to 2.67 10 5 cm2 s 1 in the galactosemic rabbit eye suggesting an increase in either free water proportion or thermal convection.

Conclusions: Resistance to water self-diffusion appeared to relate to lens fiber orientation and intracellular protein order. Diffusion imaging therefore can be used to examine water self-diffusion to detect early osmotic alteration of lens fibers.

Copyright © 2002 S. Karger AG, Basel

Introduction

Magnetic resonance imaging (MRI) can be used to study water self-diffusion [see, for example, 1]. It also has been applied to the study of biological tissues

such as the skeletal muscle [2–5]. Echo-planar-based clinical imaging has been performed on the brain [6–12], and microimaging of water diffusion has been done on rat myocardium [13], mouse kidney [14], and frog sciatic nerve [15]. A resistance to water mobility is characterized by a reduction in the diffusion coefficient (D), which is a result of water encountering membrane or subcellular barriers. Similar to the muscle and the nerve, the cortical cells of the crystalline lens are also long fibers organized in a parallel albeit a curved pattern; their ends are joined both anteriorly and posteriorly at the lens sutures. The fibers overlay the inner nucleus in an onion-like multilayer manner. In theory, a diffusion study can provide information on the organization of these fibers. Previous studies indeed have demonstrated a resistance to water diffusion between lens fibers in the rabbit eye in vitro [16]; however, it is unclear if these observations were complicated by postmortem changes and if in vivo imaging is at all possible. Indeed, there are technical difficulties in performing diffusion-weighted imaging on the lens in living animals. First, it is not possible to position all rabbits and orient their eyes identically or position the same rabbit in an exactly reproducible manner; and second, it is not possible to totally eliminate the rabbit’s motion from ocular, cardiac, and respiratory activities. In vivo studies therefore must operate under these constraints. In this report, studies on normal rabbits and two separate sugar cataract models, alloxan diabetes and galactosemia, were performed. Information on the movement of water was extracted from the images and the results reported here.

Materials and Methods

New Zealand White rabbits were used. They weighed between 1.5 and 3 kg. For diabetic induction, the rabbits were injected with 130 mg/kg of alloxan (in 0.01 M citrate buffer, pH 5) through the ear vein. Rabbits with blood glucose levels of 250–400 mg/dl 6–7 days after alloxan injection were considered diabetic (confirmed with test paper Dextrostix from Miles, Elkhardt, Ind.). Galactosemia was induced simply by feeding rabbit chow enriched with 50% galactose. All experiments were done after 2 weeks of diabetes/galactosemia. After MRI, the rabbits were sacrificed and the lenses extracted for biomicroscopic inspection. None of the lenses showed opacities.

Diffusion-weighted MRI was performed on a 4.7-tesla General Electric CSI animal system. The rabbit was anesthetized with intramuscular injection of ketamine (25–40 mg/kg)- xylazine (5–10 mg/kg) mixture through a catheter into the muscular part of the hind leg. It was then placed on its side on a Lucite cradle with one eye facing up. The head was held firmly with foam cushions and adhesive tapes. The short axis of the lens was aligned as closely as possible with the x-axis (z-axis direction of the magnetic field). A home-built 1-inch three-turn surface coil was placed directly on top of the eye to cover the entire anterior segment. Tuning and matching were first done outside of the magnet and again after the animal was in position inside the magnet. Shimming was done until maximal water-proton signal gain. A preliminary multislice scan to localize the central slice of the lens used a T1-weighted

spin-echo sequence with TR/TE 700/20 ms, a field of view of 5 cm, a matrix of 256 128 and slice thickness of 2 mm. Unlike in the in vitro study [16], several attempts were necessary to locate the central lens slice in vivo; the endpoint was reached when the slice with the largest lens dimensions was found. Once this slice was located, diffusion imaging could then proceed. If any motion artifacts were detected during imaging, the animal was removed, reanesthetized, repositioned and the whole process began again. Or a new animal was used instead.

We modified the pulsed-gradient spin-echo sequence [17] for imaging [see, for example, 13, 14, 16], in which a pair of gradient pulses were applied before and after the 180° radiofrequency pulse of a spin-echo sequence. The MRI parameters included the following: TR (repetition time)/TE (echo time) 2,000/70 ms; field of view 4 cm with a 256 128 matrix; pulse-gradient strengths (g): 0–6 Gs/cm in primary (x, y and z) and secondary coordinates (x y, x z, and y z); 20 ms (the gradient pulse duration) and 25 ms (the beginning of first gradient pulse to the beginning of the second gradient pulse). Calculation of the diffusion coefficient (D) was based on the following:

ln[A(g)/A(0)] – ( g)2 [ – ( /3)]D

where A(g) and A(0) are signals with and without gradient g, respectively, and is the gyromagnetic ratio. A plot of ln[A(g)/A(0)] as a function of ( g)2 [ – ( /3)] gives the slope D.

Calculation of increase in the equatorial zone was based on images with 0 pulsed gradient. The distance between the tip of the hyperintense equator to the outer edge of the signal-void nuclear zone was measured and calculated as percent of the long axis of the lens.

Results

The crystalline lens has very short relaxation times, its T2 is regionally dependent on the order of 10–25 ms [18]. In MRI, short T2s do not permit echo generation because the available TEs on low-field imagers are usually around 15–20 ms. As a result, the lens nucleus shows as a signal-void region. The cortex on the other hand behaves normally as other tissues, i.e., its image intensity is a function of proton relaxation times and density. On the other hand, increasing signals at a fixed TE (in the present case, 20 ms) indicates a lengthening of T2. With 0 pulsed gradient, the pulsed-gradient spin-echo sequence is essentially T2-weighted. We have noted increasing cortical areas that now produce MR signals (fig. 1). The results of the proportion of equatorial cortex in relation to the long axis of the lens are tabulated and shown in table 1. It is clear that lenses in the diabetic and galactosemic rabbits contain the largest signal-producing equators.

Diffusion-weighted imaging of the lens was done on normal, diabetic and galactosemic rabbits. In general, there is a resistance to water self-diffusion in the lens cortex: A very narrow almost undetectable cortical region (the superficial cortex) is seen with zero gradient (fig. 1) and its intensity increased when the field gradient was turned on in perpendicular to the orientation of the lens fibers (fig. 1).

The diffusion coefficient (D) was calculated from images acquired with 0, 2, 4 and 6 Gs/cm gradients (see Materials and Methods). The D values

Fig. 1. Diffusion MR images of the rabbit lens in vivo. Rabbit eye in situ is shown (a) and the eyes are highlighted (b). Note the lack of signals in the lens in the control (no diffusion gradients) and the hyperintense cortex when the gradients are turned on. The short (anteroposterior) and long axes of the lens were aligned with the x- and y-gradient, respectively, and z-axis direction of the magnetic field. Diff Diffusion; Grad gradient.

(D 10 5 cm2 s 1) are shown in table 2. The x-gradient is along the anteroposterior (the short) axis of the lens, the y-axis is along the long axis of the lens, and z is the third cardinal axis perpendicular to the anteroposterior plane. Water diffusion appears direction-independent in the aqueous humor (table 2).