British Journal of Radiology (2004) 77, 49-51
© 2004 British Institute of Radiology
doi: 10.1259/bjr/22916297
Marked increase in the rate of ocular lens regeneration in the newt, Cynops pyrrhogaster, following partial body exposure to low dose X-rays
T Okamoto, BSci,
T Kanao, BSci,
Y Miyachi, PhD and
N Nohara, PhD
Department of Radiological Sciences, International University of Health and Welfare, Kitakanemaru 2600-1, Ohtawara-shi, Tochigi-ken 324-8501, Japan
Correspondence: Dr Y Miyachi
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Abstract
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In recent years, concern over the stimulating effects of low-dose X-rays has been growing. Therefore, the effects of low-dose X-irradiation on lens regeneration in the newt were examined. Newts were subjected to sham or whole-body X-ray exposure at a dose of 0.2 Gy or 0.4 Gy, delivered at a rate of 0.43 Gy min-1. The eyeballs were fixed in formalin solution, embedded in paraffin and assessed histologically. On day 14 after lens removal, unexposed animals showed the formation of a hollow epithelial vesicle of depigmented cells continuous with the laminae of the iris corresponding to the expected regeneration stage (Reyer's regeneration stage II). In contrast, lenses from newts exposed to a 0.2 Gy dose of X-rays showed some formation of the primary lens fibre nucleus corresponding to the fibre differentiation stage (Reyer's regeneration stage III-early). Thus, low-dose X-irradiation induced regeneration compared with the unexposed groups. An acceleration from Reyer's stage II to III-early was also found on day 14 following irradiation of only the upper belly, including the spleen. The effects of low-dose X-irradiation on lens regeneration may be mediated by changes in immune activity.
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Introduction
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Among the vertebrates, the newt provides the best model for the study of lens regeneration. Able to regenerate limbs, tail, parts of the brain and the eyes, the newt shows a greater capacity for regeneration of body parts than any other vertebrate. Wolffian's experiments studying lens regeneration in the newt show that, during lens regeneration, fully differentiated iris epithelial cells located in the mid-dorsal margin of the iris epithelium are converted into lens cells after passing through two or more induced cell divisions. During this transformation, activated by lentectomy, the cells go through the following stages: 1) induction of DNA replication and mitosis; 2) depigmentation; and 3) formation of the lens fibre nucleus [1, 2].
Recently, concern over the biological effects of low dose X-rays has been growing. "Hormesis" is the name given to the stimulatory effects of small doses of substances that in larger doses are inhibitory. This term may also be applied to any physiological effect that occurs at low doses that cannot be anticipated by extrapolating from the toxic effects noted at high doses. Exposure to low doses of X-rays, for example, reportedly stimulate antioxidant defense, membrane signal transduction, inflammation and immune reactivity [3–5]. In this study, we investigated the effect of low-dose X-rays on lens regeneration.
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Materials and methods
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Adult newts, Cynops pyrrhogaster, were reared in polycarbonate cages in an environmentally controlled room (temperature: 22°C±1°C; humidity: 60%±10%), with a standard 12 h light/dark cycle. The animals were subjected to X-rays at a dose of 0.2 Gy or 0.4 Gy with an X-ray machine (Toshiba-KX, Japan), given at a rate of 0.43 Gy min-1 (125 kV, 3 mA, 0.3 mm Cu+ 0.5 mm Al filter). Output was measured by a Victoreen (USA) ionization chamber. In the first set of experiments, animals were exposed to X-rays 3 days after lentectomy. Eyeballs were removed on days 14 or 21, serial histological sections were prepared and the extent of lens regeneration was evaluated. In the second set of experiments, animals were irradiated 3 days prior to lentectomy and eyeballs were evaluated on day 14. In a third set of experiments, only the upper-belly region of the animals was exposed to X-rays. The rest of the body was shielded from the X-rays by inserting the animals into 10 ml injectors covered with a 2 cm thick lead shielding. A lead plate reduced the radiation dose to zero. Animals were not anaesthetized during the irradiation phase. Sham-irradiated control animals were subjected to identical procedures in the X-ray machine, but were not actually exposed to the X-rays.
Sato [2] designated 13 stages in lens regeneration that were subsequently used by Reyer [1]. The histological procedure adopted in the present study was similar to that described in Reyer [1]. The eyeballs were surgically removed 7 days or 14 days after lentectomy and fixed for 48 h in glutaraldehyde buffered with 0.15 M sodium phosphate at pH 7.3. They were then washed in several changes of the same buffer and embedded in paraffin. Serial sections (2 µm thick) were prepared and stained with Mayer's haematoxylin and eosin.
Statistical analysis of all experimental data was performed by a Chi-square test.
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Results
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Effects of whole-body X-irradiation on lens regeneration
In unexposed animals, 14 days after lentectomy, formation of a hollow epithelial vesicle of high cuboidal depigmented cells continuous with the epithelia of the outer and inner laminae of the iris was observed. This structure corresponds to the expected regeneration stage (Reyer [1], regeneration stage II). In contrast, animals exposed to a 0.2 Gy X-ray dose 3 days after lentectomy showed the beginning of the primary lens fibre nucleus projecting from the inner wall into the lumen of the lens vesicle as a little hillock. This structure corresponds to regeneration stage III-early (Figure 1
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Figure 1. Changes in regeneration processes of the newt's lens on day 14 following low-dose X-irradiation. (a) A lens of unexposed animal (Reyer's regeneration stage II). (b) 0.2 Gy exposed animal (Reyer's regeneration stage III-early).
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Similar radiation effects were obtained when the time interval between lentectomy and sectioning was extended to 21 days (Table 1). Eyeballs of unexposed newts showed primary lens fibre differentiation consistent with Reyer's regeneration stage III-early. In contrast, eyeballs from newts exposed to a 0.2 Gy dose of X-rays showed secondary lens fibre differentiation beginning at the equatorial zone of the lens epithelium, where it meets the primary lens fibre nucleus. The secondary lens fibres surrounded the primary lens fibre nucleus, except laterally, corresponding to regeneration stage III-late. Increasing the radiation dose to 0.4 Gy resulted in the complete disappearance of the radiation-induced effects on lens regeneration (Table 1). Together, these results indicate that lower dose level induces differentiation of lens tissue.
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Table 1. Acceleration of the rate of lens regeneration following partial-body, upper-belly exposure to low-dose X-rays
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The effect of timing of the lentectomy relative to irradiation was then examined. When newts were exposed to low-dose X-rays on day 3 prior to the lentectomy no differences in lens regeneration between animals exposed to low-dose irradiation and sham exposed controls were observed (Table 1
). On day 14, both sham and 0.2 Gy exposed animals exhibited characteristics of regeneration stage II. These data indicate that the radiation-induced effect is observed in newts exposed to X-rays after lens removal but there are no effects of X-rays on lens regeneration if the animals are exposed to X-rays prior to lens removal.
Acceleration of the rate of lens regeneration following partial body exposure to low-dose X-rays
Changes in lens regeneration following upper belly (including the spleen) exposure to low-dose X-rays are shown in Table 1. Eyeballs were evaluated on day 14 in an experiment in which newts were irradiated after lentectomy. An acceleration from regeneration stage II to III-early was observed while no such effect was found in sham-irradiated, control animals. These data indicate that upper belly irradiation is sufficient to produce the same effects as whole-body irradiation. These effects might be related to changes in immune activity.
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Discussion
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The data in the present study show that whole-body X-irradiation with 0.2 Gy after lentectomy promotes lens regeneration. However, increasing the radiation dose to 0.4 Gy results in the complete disappearance of the radiation-induced effects on lens regeneration. In general, the biological effects of radiation are positively correlated with the amount of exposure. However, our results suggest that this relationship does not apply to the effects of irradiation on lens regeneration.
A similar dose–response relationship is emerging for the cellular effects of oxidant exposure. In contrast to the well-documented cytotoxic effects of exposure to high levels of oxidants, evidence is accumulating that exposure to extremely low doses of oxidants may have a stimulatory effect on various cellular processes [3, 4]. Many reports have suggested that low-dose X-irradiation enhances the immune function of T-lymphocytes and splenocytes. Our data indicate that irradiation of only the upper belly, including the spleen, results in an acceleration of lens regeneration (Table 1). Therefore, it is possible that this augmentation in lens regeneration is mediated by subtle changes in splenic immune activity following low-dose X-irradiation.
The visible alterations in the dorsal iris that mark the beginning of lens regeneration appear a few days after lens extirpation. What is the initiating factor of lens regeneration? Uhlenhuth [6] reported that depigmentation and subsequent lens formation was brought about by the rupture of the iris membrane during lens extirpation and consequent exposure of the dorsal iris cells to the fluids of the eye chamber. Our data show that regeneration was not accelerated if animals were irradiated prior to lentectomy (Table 1). This suggests that the primary inflammatory reaction induced by lentectomy may be a necessary prerequisite for induction of the radiation effects on lens differentiation. Many studies indicate that small incisions made following low-dose radium exposure in rabbits and dogs result in faster healing compared with unexposed controls; high doses of radium retarded wound healing. There also is growing evidence that increased activity of immune system would promote wound healing in the iris [7, 8]. These findings are consistent with our findings since regeneration was accelerated only when animals were irradiated after lentectomy and associated inflammation. In summary, our data suggest that exposure to low-dose X-irradiation may stimulate the biological defense system.
Received for publication October 15, 2002.
Revision received June 9, 2003.
Accepted for publication September 15, 2003.
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References
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- Sato T. Vergeleichende studien uber die geschwindigkeit der wolffschen linsen regeneration bei Triton Taeniatus und bei Diemyctylus pyrrhogaster. W Roux' Arch f Entw'mech 1940;140:570–613.[CrossRef]
- Miyachi Y. Acute mild hypothermia caused by a low dose of X-irradiation induces a protective effect against mid-lethal doses of X-rays, and a low level concentration of ozone may act as a radiomimetic. Br J Radiol 2000;73:298–304.[Abstract]
- Oliveieri G, Bodycote J, Wolff S. Adaptation response of human lymphocytes to low concentration of radioactive thymidine. Science 1984;223:594–7.[Abstract/Free Full Text]
- Kelsey KT, Memisoglu YA, Frenkel D, Liber HL. Human lymphocytes exposed to low doses of X-rays are less susceptible to radiation-induced mutagenesis. Mutat Res 1991;263:197–201.[CrossRef][Medline]
- Uhlenhuth E. Studien zur linsen regeneration bei den Amphibien. I. Arch EntwMech Org 1920;46:149–68.
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Abstract
Introduction
