International Journal of Radiation Biology
ISSN: 0955-3002 (Print) 1362-3095 (Online) Journal homepage: http://www.tandfonline.com/loi/irab20
Effect of Ionizing Radiation on Neuromuscular Junctions in Mouse Tongues R. Gorodetsky, G. Amir & R. Yarom To cite this article: R. Gorodetsky, G. Amir & R. Yarom (1992) Effect of Ionizing Radiation on Neuromuscular Junctions in Mouse Tongues, International Journal of Radiation Biology, 61:4, 539-544, DOI: 10.1080/09553009214551301 To link to this article: http://dx.doi.org/10.1080/09553009214551301
Published online: 03 Jul 2009.
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RADIAT. BIOL .,
Effect of ionizing radiation on neuromuscular junctions in mouse tongues R. GORODETSKY*t, G . AMIR$ and R . YAROM$
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(Received 10 May 1991 ; revision received 9 September 1991; accepted 2 October 1991)
Abstract . Radiation damage to the neuromuscular junctions (NMJs) in mouse tongues was studied using local x-irradiation of the tongues with the rest of the body shielded . Transmission electron microscopy (TEM) revealed no significant morphological changes in the fine structures and organelles of the NMJs given 4 Gy. A dose of 8 Gy produced degenevative morphological changes associated with oxon terminal sprouting as early as 2 and 7 days following irradiation . Subsequently, 111 weeks later, severe degenerative changes were observed . The number of mitochondria was significantly decreased with increased occurrence of degenerative membranal features . The number of synaptic footplates without terminals or with multiple small terminals within one groove increased gradually with time. Most of these pathological changes persisted for at least 3 months after irradiation . However, the myofibres, blood vessels and interstitial cells appeared to be unaffected throughout the period of follow-up . The present study substantiates our previous reports of ageing-like changes in the tongues' NMJs induced by their excessive exposure to free radicals .
1. Introduction The effect of ionizing radiation on the function and morphology of neuromuscular junctions (NMJs) in mammals has not been investigated in detail (Love and Gomez, 1984) . Studies of the damage inflicted by high doses of radiation to nerve cells in different parts of the central nervous system (CNS) are more common . (Carpenter et al . 1986, Masuda et al . 1977, Lian 1989) . These effects were typically expressed by impaired function of the target organ, by delayed paralysis or by the release of molecular markers indicating specific damage to different cell types of the CNS, Martins et al. 1979, Fisher et al . 1987, Tiller-Borchich et al. 1989) . None of these reports considered the specific contribution of the radiation damage inflicted on the NMJs. Ultrastructural histology was employed only for the evaluation of the regeneration of injured axons exposed to high doses of radiation (Love 1983, Love et al . 1986) . *Author for correspondence, tSharett Institute of Oncology and Department of Pathology, Hadassah University Hospital and Medical School, Jerusalem, Israel .
The tongue muscle, in relation to typical skeletal musculature, has an extremely high density of NMJs . This enables a detailed histological and morphometric study to be made using transmission electron microscopy (TEM), as previously reported by Fahim & Robbins (1982) and by Cardasis and LaFontaine (1987) . Yarom et al. (1986, 1987) have previously used this procedure to study free radicalinduced damage to the NMJs in tongues of young patients with Down's syndrome (DS), where premature ageing-like changes in these structures were observed . The changes in the NMJs were speculated to be the result of an elevation in the level of free radicals generated by over-expression of Cu-Zn superoxide dismutase (SOD) (Sinet, 1982) . Similar changes were also observed by Yarom et al. (1988) and by Avraham et al. (1988) in the NMJs of transgenic mice with an extra Cu-Zn SOD gene . The present study, employing small field x-ray irradiation of the tongue with the rest of the body shielded, may contribute to the better understanding of free radical damage confined to the NMJs .
2. Materials and methods 2.1 . Animals Twenty-two Balb-C mice (weighing 18-22 g), 8-11 weeks old at the beginning of the experiments, were used . All were less than 6 months old at sacrifice . They had free access to food and water and were treated in accordance with the guidelines for humane treatment of experimental animals . For the tongue irradiation the mice were anaesthetized by intraperitoneal injections of 60 mg/kg sodium pentobarbital. They were then placed lying on the side in a special jig with the hind legs restricted by a rubber band bound backwards . Another rubber band over the upper part of the body helped immobilize the anaesthetized animals during irradiation . The jaws and mouth were kept open by separating the upper and lower incisors with
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R. Gorodetsky et al .
two upright thin rods, 5 mm apart . The whole body of the mouse was protected by a 2 mm thick lead shield . The tongues were exposed to small field xirradiation by using a small wedge-shaped opening in the shield which allowed the x-rays to reach more than 75% of the anterior part of the exposed tongue and part of the lips . Thus, the muscle and the NMJs were irradiated while the neurons and their cranial nuclei were not exposed . X-ray doses of 4 Gy (four mice) and 8 Gy (18 mice) were given to the exposed tongue using a 175 kVp/20 mA Philips machine, with 2 mm Cu filter, at a dose-rate of 0 .95 Gy/min . The average dose given to the middle of the tongue was calibrated using a thin ionization chamber placed in tissue-equivalent material in conditions resembling those of the experiment . Due to the small field, variations in the actual dose delivered of up to 6% occurred . The four control mice were also anaesthetized and were given sham-irradiation . The mice were sacrificed by cervical dislocation at 1 and 2 days, 1 and 2 weeks, 1, 2 and 3 months after the irradiation (two to four animals at each time) . The four animals given 4 Gy were killed at 1 and 2 months after irradiation .
2.2 . Preparation of tongues of electron microscopy The tongue of the sacrificed mouse was rapidly excised and its anterior part was immersed into 2.5% glutaraldehyde buffered with 0 . 1 M sodium cacodylate . The muscle was trimmed to remove the mucosa, cut into smaller pieces and left in fixative for 2 h . After overnight rinsing in buffer and postfixation in cacodylate-buffered osmium tetroxide the specimens were dehydrated and embedded in Araldite . Semi-thin sections stained with toluidine blue were examined by light microscopy . Sections containing nerve endings were cut for electron microscopy (two to six blocks from each tongue) . They were stained with uranyl acetate and lead citrate, and examned in a Philips 300 transmission electron microscope .
tablet with Summa-graphics ID-1 operated in a DEC PDP-11 /34 computer . Qualitative observations related to the number of terminals per junction, or to the manifestation of structures associated with the degeneration of the NMJ, such as membranous whorls, vacuoles and filaments, were recorded in each point of analysis as the percentage of NMJs in which they occurred out of the total number examined . Quantitative measurements included : nerve terminal area (in µm2) ; number and volume fraction (V,,) of mitochondria in terminal axons . In some cases the synaptic cleft width, the number of secondary folds and microvesicle density were also noted . A two tailed Student's t-test was used for statistical comparisons of quantitative data . For the qualitative changes a chi-square test was used . 3. Results In general, the mice behaved, ate and drank in a normal fashion and seemed unaffected by the exposure of their tongue to either a dose of 4 or 8 Gy . In detailed histological examination by TEM, the NMJs of mice irradiated with 4 Gy and sacrificed 1 and 2 months later did not show any significant deviation from normal controls with respect to time points with sham irradiation (Figure 1) . Therefore, the results from these time points from both groups were combined (Table 1)] . The mice irradiated with 8 Gy revealed marked pathological changes in their NMJs (Table 2) in comparison to the grouped results obtained from the
2.3 . Morphometry In each tongue seven to 16 separate NMJs were photographed on a high-resolution 35 mm film, usually with a magnification of x4000 . The final magnification presented was within the range of 12 000-40 000 . Area measurements, as well as point intersection counts, were done with a manual cursor on enlarged projections of the films onto a digitizing
Figure 1 . A neuromuscular junction (NMJ) from the tongue of a mouse 1 month after 4 Gy (magnification x 12 000) . The nerve (N), terminal axon (A) and myofibres (M) are normal in appearance .
Radiation effects on neuromuscular junctions
Table 1 .
Morphometry of NMJs exposed to 4 Gy .
No. of NMJs (and animals) Area of terminal axons (µm 2 ) No . of mitochondria per terminal axon Mitochondrial volume fraction (V,.) Percentage NMJs with degenerative structures Percentage NMJs without terminals Percentae NMJs with multiple terminals
1 and 2 months following 4Gy irradiation
30 (3) 5 . 3±1 . 8 11 . 5±3 . 9 0 . 29±0 . 14 15 8 10
38 (4) 7 .2±3 . 5 12 . 7±7 .1 0 . 26±0 .09 14 5 5
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The results of the observations 1 and 2 months following irradiation did not differ significantly and were therefore cumulated. Errors represent the standard deviations of the mean .
controls which were examined 1, 2, 4 and 8 weeks after sham-irradiation . Twenty-four hours after 8 Gy, the NMJs usually appeared undisturbed morphologically . Two days after irradiation many NMJs showed pathological changes consisting of irregularity of terminal axons with the presence of membranous whorls, vacuoles, tubules and filaments (Figure 2) . Also small 'out-pouching' of the terminals was observed (Figure 3) or axonal sprouting which contained microvesicles with only a few mitochondria . In a few NMJs the terminal axolemma as well as the sarcolemma of the secondary folds seemed to be degenerating (Figure 2) . The microvesicles were as a rule abundant and the mitochondria appeared to be undisturbed . One week after irradiation most of the terminal axons still showed qualitative signs of degenerated membranes, vacuoles, tubules and filaments (Figure 4) . The mitochondria significantly decreased in number, but their total volume fraction decreased Table 2 .
less dramatically (Table 2), suggesting the possibility of an increase in the mean size of the remaining mitochondria. Two weeks after irradiation a persistent decrease in the number of mitochondria, as well as an increase in the occurrence of degenerative structures, were observed . The primary grooves were sometimes flattened or disorganized . There seemed to be an increase in the occurrence of grooves without terminals on one hand, or multiple small terminals on the other (Table 2) . The number of secondary folds showed more variability, but on the whole they were not significantly different from controls. The synaptic cleft width was often wider than in controls but was difficult to measure accurately. The NMJ pathology seen at that stage was preserved showing no recovery of the radiation-induced damage as long as 1, 2 and even 3 months after irradiation (Figures 5 and 6) . During the whole period of follow-up, the myo-
Morphometry of neuromuscular junction at various times after X-irradiation with 8 Gy. Controls
No . of NMJs (and animals) Area of terminal axon (µm 2 ) Synapse width (µm) No . of mitochondria per terminal axon
44(4) 5 . 9±3 .5 45+12
18 (2) 5 . 4±3 . 1 n .d .
14 (2) 5 . 8±2 . 8 n .d .
26 (4) 4-3+2-8 n .d .
70 (4) 3.2 ± 2 . 0 n .d .
50 (3) 3-4± 2 . 5 77±7
28 (3) 3-3+2-9 74±19
21 (2) 3-2±1-6 81±21
6 . 4±4 .4
5 . 9±4 . 6
6. 1±8 . 4
7 . 1±7 . 3
7 . 5±5 •I
Volume fraction (V,,) of mitochondria
0-31+0-12 0.29±0 .14 0 . 28±0 . 18 0 . 25±0 . 14 0 . 22±0 . 11 0 . 15±0 . 13 0 .20±0.18 0.14±0 .07
Percentage of NMJs with degenerative structures Percentage of NMJs without a terminal axon Percentage of NMJs with multiple terminals
All the values beyond 2 weeks are significantly different from controls (p