Radiotherapy and Oncology, 25 (1992) 295-300 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0167-8140/92/$05.00
295
RADION 01028
Fractionation sensitivity of the rat cervical spinal cord during radiation retreatment A. C. C. Ruifrok, B. J. Kleiboer and A. J. v a n der Kogel Institute of Radiotherapy, University of Nijmegen, Nijmegen, The Netherlands (Received 4 February 1992, revision received 7 April 1992, accepted 21 April 1992)
Key words: Spinal cord; Radiation myelopathy, Long-term recovery; Retreatment, Fractionation; Late effect
Summary Data concerning the fractionation sensitivity of normal tissues during radiation retreatment are limited. Experiments were performed to investigate whether the fractionation sensitivity of the rat cervical spinal cord is changed during retreatment 6 months after a first dose of 15 Gy, representing about half the biologically effective dose for induction of paresis. After a 6 months interval, the long-term recovery from the first treatment was about 45 %. The fractionation sensitivity of the rat cervical spinal cord during reirradiation was not significantly different from the fractionation sensitivity of not previously irradiated control rats, with an ~/flratio of 2.3 Gy in control rats and 1.9 Gy during reirradiation of the spinal cord. An additional observation from these experiments was the presence of incomplete repair after fractionated treatment with 2 fractions of 3 Gy per day with 10-h intervals.
Introduction For several acute as well as late reacting normal tissues, animal studies concerning retreatment tolerance have been performed. Most of the work concerning longterm recovery has been directed to the time course and extent of recovery after single dose or fractionated first treatments and single dose test treatments [8,9,12,13,17,19-22,30,33,35]. Data concerning the fractionation sensitivity during retreatment are limited. Stewart et al. [ 18] showed that in mouse kidney there was no reduction in fractionation sensitivity when fractionated irradiation was given to mice irradiated 26 weeks before. However, Turesson and Thames [28] reported an increase in ~/fl ratio as a function of overall treatment time during treatments of more than 4 weeks for the early reactions of erythema and desquamation of human skin, suggesting a decreased fractionation sensitivity, while no clear change in ~/fl for the late reaction of telanglectasia was found [28]. Although prolonged treatments are not directly comparable to
reirradiation, the data indicate that in some situations the fractionation sensitivity of a tissue may change depending on previous radiation treatments. This may have important consequences for the normal tissue tolerance and the optimal fractionation schemes for retreatment in case of tumor recurrences in previously irradiated sites. The present experiments were performed to investigate whether the fractionation sensitivity of the rat spinal cord is changed during radiation retreatment.
Methods and materials Female Wistar rats ( C P B / W U ) were used in this study at an age of 14-20 weeks (adult rats). The rats were housed in macrolon cages and provided with water and food ad libitum. Prior to irradiation, the animals were anesthetized with Ethrane inhalation [5]. Positioning was facilitated using a lucite fixation setup, enabling us to irradiate six animals simultaneously. Irradiations were performed on a linear accelerator
Address for correspondence: A. J. van der Kogel, Institute of Radiotherapy, University of Nijraegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.
296 at a focus-skin distance of 100 cm. Exposure of a 18 mm segment of the spinal cord (C1 through T1-T2) was carried out with 4 MeV photons at a dose rate of 2.2 Gy/min. The head and body were shielded with 70 mm lead blocks close to the skin. All rats received a first irradiation dose of 15 Gy. A second course of irradiation was given either at one day (control), or at 6 months after this first dose, with "single dose" or fractionated treatments, with fraction sizes of 3, 4 and 6 Gy. The 4 and 6 Gy fraction treatments were given 5 days per week; the 3 Gy per fraction treatments were given 5 days per week with 2 fractions per day with a 10-h interval. Also a control treatment with one fraction of 3 Gy per day was performed. Each experiment comprised 5-6 dose levels with 5-6 animals per dose group. Examinations for signs of neurological impairment were performed 3 times a week, starting 3 months after the last treatment course. Animals were scored as responders when they showed regular dragging of their feet with palmar flexion, dragging of extended forelegs, or inability to walk on their forelegs when lifted by the tail. When these definite signs of paresis were seen, rats were sacrificed. Follow-up of the animals was continued up to 300 days after the last irradiation. The sacrificed animals were perfused with saline and 4~o buffered formaldehyde solution with a standard infusion set at a pressure of 100-120 cm H20. The irradiated part of the spinal column was dissected out for histological examination. The spinal columns were decalcified in 20~o formic acid, 4 ~ sodium formate solution, and embedded in paraffin. Haematoxylineosin stained sections of 6 #m thickness were routinely examined for histological damage. Rats of which the neurological symptoms could be attributed to spinal cord compression by a tumor, or damage to vertebrae resulting in spinal cord compression, were not recorded as responders but as intercurrent deaths. Response rates were corrected with the life-table method when intercurrent deaths occurred [1,15]. Dose-response curves were constructed by probitanalysis. Significance of differences in doses resulting in 50~o probability of paresis (50~o effective dose, EDso) were tested using the Z2-test [ 14]. Fitting of the linear quadratic (LQ) model to the data was done with the direct data analysis method of Thames et al. [26] using a computer program provided by the Department of Biomathematics in Houston [7]. Results
Irradiation of the cervical spinal cord of the rats resulted in paresis of the forelegs after latent periods of
TABLE I Latent times for development of paresis after a first dose of 15 Gy, followed by fractionated irradiation after one day or 6 months, at an isoeffective range of EDso-ED99. Treatment
Latency after first fraction* (days) mean _+SD (median)
15 G y + S D 15 Gy + 6 Gy/fx 15 G y + 4 Gy/fx 15 G y + 3 Gy/fx; 2 fx/day
Interval one day
Interval 6 months
233+18(235) 187 + 14 (192) 199+25 (196) 218_+25 (221)
160+21(162) 152 + 23 (150) 156_+25 (146) 165+60(151)
* Overall treatment time for fractionated treatments with a second course after one day was 8-18 days, for fractionated retreatments after 6 months 11-29 days.
5 to 7 months. In Table I, the latent periods after treatments associated with 80-99~o probability of paresis (EDso-ED99), are summarized. Following control treatments the latency tends to increase with decreasing fraction size, however, this increase is not significant due to the large spread in latency times after the treatments. Second course irradiation with a single dose even results in a longer latent time than observed after treatment with the largest fraction size of 6 Gy. Following irradiation 6 months after the first dose all latent times are shortened by about 1-2 months compared to the latency after control treatments. No indication of fraction size dependent latency is observed after a 6 months interval. Figure 1 shows the probit curves for development of paresis during the follow-up period of 300 days after
% paretic
loo%
•
,t
80%"
/
60%
!
m
•
40% 20%" 0%
, 0
20
40
610
8Uo
1 O0
D o s e (Gy)
Fig. 1. Dose-response curves for paresis after tionated control treatments one day after a --.--, SD; ---------I~, 6 Gy/fx; - - & - - , 3 Gy/fx, 1 fx/day; .... [] .... ,3 Gy/fx, 2 fx/day. fidence interval.
single dose and fracfirst dose of 15 Gy. 4 Gy/fx; - - U - - , Error bars: 95~ con-
297 "single dose" and fractionated control treatments. The solid lines represent "single dose" and fractionated treatments given with time intervals of at least 24 h; the dotted line represents the result of fractionated treatment with 2 fractions of 3 Gy per day with a time interval of 10 h. The EDso values calculated from these probit curves are summarized in Table II. As can be seen, the EDso increases with decreasing fraction size when the time interval between fractions was at least 24 h. Treatment with 2 fractions of 3 Gy per day resuited in a significantly lower EDso of 42.0 Gy compared to the EDso of 56.6 Gy after treatment with 1 fraction per day (p < 0.05). In Fig. 2, the probit curves for single dose and fractionated reirradiation 6 months after the first dose of 15 Gy are shown. The solid lines represent the single dose and one fraction per day treatments. The dotted line represents the treatment with 2 fractions of 3 Gy per day with an interval time of 10 h. The EDso values
TABLE II EDso values for paresis after cervical spnal cord irradiation with a first dose of 15 Gy, followed by a second irradiation course after one day or 6 months. Treatment
15 15 15 15 15
EDso (Gy) of the second irradiation course (95 % confidence interval)
Gy + SD Gy + 6 Gy/fx Gy + 4 Gy/fx G y + 3 Gy/fx; 1 fx/day Gy + 3 Gy/fx; 2 fx/day
Interval one day
Interval 6 months
16.2 (15.5-17.3) 36.8 (34.2-40.4) 47.2 (44.4-49.9) 56.6 (50.2-98.4) 42.0 (35.2-49.3)
18.5 (17.5-19.7) 43.2 (30.7-50.1) 65.1 (57.1-75.0) 54.9 (45.4-60.9)
% paretic
00,
y y
80%"
/j-
40%"
:,'£]
0%
, ,w 0
I , 20
610 (Gy)
Function paramter values of direct data analysis of rats irradiated one day and 6 months after 15 Gy priming irradiation treatment. Parameter
InK 10.~ (Gy- 1) 100.fl(Gy -2) ~/fl(Gy)
Function parameters (95% confidence interval) One day interval
6 months interval
5.8 0.47 2,1 2.3
5.2 0.29 1.5 1.9
(4.5-7.2) (0.32-0.62) (1.5-2.6) (1.5-3.3)
(3.4-7.0) (0.13-0.46) (1.0-2.1) (0.7-3.8)
Function: E = - InK + otD + ~D21N, with E = - log survival for isoeffect, K the number of "tissue rescuing units", ~ and fl the parameters of the LQ model, D the total dose and N the number of fractions [26].
calculated from these curves are summarized in Table II. Also after reirradiation 6 months after the first dose of 15 Gy the EDso increases with decreasing fraction size when daily fractions were given; however, treatment with 2 fractions of 3 Gy per day resulted in an ED50 of about 55 Gy, which is considerably lower than the ED50 of about 75 Gy expected on the basis of the LQ model with complete repair between the fractions. To estimate the fractionation sensitivity of control rats and rats treated 6 months after the first dose of 15 Gy, the paresis data were analyzed using the direct analysis method of Thames et al. [26]. Because the results of the experiments with 2 fractions of 3 Gy per day were different from the predictions on the basis of the LQ model, the calculations were performed using the data of "single dose" and daily fractionation treatments only. The results of these calculations are summarized in Table III. Although the results suggest a minor decrease in the absolute values of ct as well as during the reirradiation treatment" after 6 months, we observed no significant difference in the ct/pratio for irradiation at one day or 6 months after a first dose of 15 Gy; the ct//~ratio is about 2 Gy after second course irradiation after one day as well as during reirradiation after 6 months.
•
Discussion
, , 40 Dose
TABLE III
810
1 00
Fig. 2. Dose-response curves for paresis after single dose and fractionated second course irradiations 6 months after a first dose of 15 Gy. - - e - - , SD; - - ~ I ~ - - , 6 Gy/fx; - - & - - , 4 Gy/fx; .... [] .... ,3 Gy/fx, 2 fx/day. Error bars: 95% confidence interval.
The latency to paresis after fractionated irradiation treatment of the spinal cord has been suggested to be fraction-size dependent [6]. Although the latency data of the experiments with a second irradiation course at one day after a single dose of 15 Gy also suggest a fraction size dependent latency, the differences in the latent times are not significant (p > 0.05) (Table I). The
298 latent times after a second irradiation course after 6 months give no suggestion of fraction size dependence at all. Therefore, the data were analyzed using direct estimation of the fractionation characteristics without latent time analysis [6,26]. The experiments presented in this paper were designed to determine the fractionation sensitivity of the rat cervical spinal cord during retreatment. Because of the expected high tolerance dose for a retreatment using 3 Gy fractions, this particular schedule was carried out with 2 fractions per day, to limit the overall time to about 4 weeks for the highest dose. The chosen interval time of 10 h between the 2 fractions given on one day was assumed to be sufficient for complete SLD repair according to reported repair half-times (ill2) of 1.4 to 1.9 h for rat spinal cord [3,4,25]. However, as shown in Fig. 2 and Table II, the 3 Gy fractionation resulted in a lower EDso than expected; the observed ED~o was even lower than after treatments with 1 fraction of 4 Gy/day. This indicated that a 10-h interval was not sufficient for complete repair in this experiment. To investigate this apparent loss of tolerance with 2 fractions per day, control treatments were performed with 1 and 2 fractions of 3 Gy/day. These latter experiments supported the notion of incomplete repair for 2 fractions per day in the retreatment experiments; the 2 fractions per day treatments resulted in a significantly ( p < 0.05) lower EDso than the single fraction per day treatment. Assuming that the difference in EDso values after 1 and 2 fractions per day is the result of incomplete repair, these data indicate a t,/: of about 5.5 h when mono-exponential decay of SLD is assumed [23]. More recently, it has been suggested that repair in the spinal cord may exhibit a two-component decay of the SLD. Hopewell and van den Aardweg [11] suggested t,/~ values of 10.6 min and 2.4 h for spinal cord on the basis of graphical analysis of the data of Ang et al. [4]. Although this method of analysis has been criticized [ 10], there is a trend in the presented data suggesting that the assumption of mono-exponential decay of SLD is too simple. Ang et al. [2] reported for the rat spinal cord t,/2 values of 0.7 and 4 h, with 6 5 ~ of the damage repaired by the long repair tl/2, when experimental data were fitted by a bi-exponential repair model. Also for the rat kidney [16] and lung [34], and pig skin [29] indications of two-component repair of SLD have been reported. The decay of SLD may be dose-dependent [4,16], show repair saturation [24], may be interval-dependent [10], or may be a two-component process [2,11,16,22,29,34]. However, as long as no sufficient
data are available to determine the exact nature of SLD repair, mono-exponential repair models usually provide an adequate description of normal tissue responses to fractionated irradiation. The present experiments were not designed to determine the kinetics of SLD repair, and are insufficient to determine repair times in a twocomponent model; nevertheless, the data suggested the presence of a long repair time, and clearly show a significant continued recovery between 10 and 24 h. Retreatment of previously irradiated cervical spinal cord after 6 months shows that the tolerance of the spinal cord is increased compared to treatment directly after the first dose. When expressed as a percentage of the total effect (TE) according to the LQ model [ 17,27], the first dose of 15 Gy represents about 45 ~ of the TE, with a remaining tolerance of 55~o TE. The reirradiation tolerance for single dose and fractionated retreatments after 6 months recovery represents about 7 5 ~ TE, indicating that the total dose that can be given in two treatment courses with 6 months interval represents about 120~o of the TE that can be given in a single treatment course. This value is similar to the long-term recovery found previously for immature rats of the same strain [17], but somewhat lower than reported before for adult Wag/Rij rats [33]. The data indicate that only about 45 ~o of the damage of the first treatment is recovered at 6 months. Also the shortening of the latent time after irradiation at 6 months compared to one day is in accordance with considerable damage from the first treatment still being present after 6 months. Because the data obtained for 2 fractions per day were clearly influenced by incomplete repair, only the 1 fraction per day was used for analysis of fractionation sensitivity directly after the first dose and during retreatment after 6 months. The results of this analysis are presented in Table III. The absolute values of the parameters ~ and fl of the LQ model seem to be slightly different for control treatments and reirradiation after 6 months; however, these values are strongly influenced by experimental variation in the original data, and the confidence intervals show that the differences are not significant. The most important result of the direct analysis is that the calculated ~/flratio during control treatment and reirradiation 6 months after 15 Gy are not significantly different, 2.3 Gy during control treatment and 1.9 Gy during a reirradiation treatment after 6 months, indicating that the capacity of SLD repair is not changed during retreatment. This means that for the rat spinal cord, like for the mouse kidney [ 17], there is no indication for reduction in capacity of SLD repair during reirradiation. The 2 fraction per day data suggest
299 that also the rate of repair in the rat spinal cord is comparably slow during control treatment and reirradiation after 6 months. The present data of the cervical spinal cord and those of the mouse kidney [ 18] show no change in the fractionation sensitivity during reirradiation. However, Turesson and Thames [28] found an increase in ~/fl for the early reactions of erythema and desquamation of human skin during prolonged treatment for more than 4 weeks, while no clear change in ~/fl for the late reaction of telangiectasia was found. These present and previously reported [ 18,28] results may indicate that in late reacting tissues, with normally a low cellular turnover, cellular kinetics may be not or only slightly changed during prolonged irradiation or reirradiation, while in acute responding tissues redistribution in the cell cycle and proliferative response may cause a change in the fractionation sensitivity. An acute proliferative response may result in a greater heterogeneity in the sensitivity of the target cells during prolonged treatment or reirradiation, resulting in a change in the ~/flvalue determined. A possible change in the fractionation sensitivity may also depend on the timing of reirradiation; the air ratio may be changed at the moment of maxi-
mal cellular responses to the first irradiation, while no significant change in ~/fl may be found before or after the maximal response. Summarizing, the present experiments indicate that: (1)the fractionation sensitivity of the cervical spinal cord during retreatment at 6 months after a first dose of about 45 ~o TE is not significantly changed compared to the fractionation sensitivity of the cervical spinal cord of control animals; (2) at 6 months after a first dose to the cervical spinal cord, representing 45 ~ TE, about 45 ~o the damage of the first dose has been recovered; and (3) in the rat cervical spinal cord significant repair continues to take place between 10 and 24 h.
Acknowledgements The author wish to thank W. F. M. Brouwer for physics support, T. Oostendorp for expert technical assistance, J. Koedam and coworkers of the Central Animal Laboratory for excellent collaboration, and H . D . Thames and C. Smith for providing the computer program for direct data analysis. This work was supported by grant N U K C 88-2 from the Dutch Cancer Society.
References 1 Abratt, R.P. Reporting the late complications of cancer. Br. J. Radiol. 56: 348-349, 1983. 2 Ang, K.K., Jiang, G.L., Guttenberger, R., Thames, H . D . , Stephens, L. C., Smith, C. D. and Feng, Y. Impact of spinal cord repair kinetics on the practice of altered fractionation schedules. Radiother. Oncol. in press. 3 Ang, K. K., Thames, H. D., van der Kogel, A. J. and van der Schueren, E. Is the rate of repair or radiation-induced sublethal damage in rat spinal cord dependent on the size of dose per fraction? Int. J. Radiat. Biol. Oncol. Phys. 13: 557-562, 1987. 4 Ang, K.K., van der Kogel, A.J., van Dam, J. and van der Schueren, E. The kinetics of repair of sublethal damage in the rat cervical spinal cord during fractionated irradiations. Radiother. Oncol. 1: 247-253, 1984. 5 Ang, K. K., van der Kogel, A. J. and van der Schueren, E. Inhalation anesthesia in experimental radiotherapy: a reliable and time-saving system for multifractionation studies in a clinical department. Int. J. Radiat. Oncol. Biol. Phys. 8: 145-148, 1982. 6 Bentzen, S. M., Thames, H. D., Travis, E. L., Ang, K. K., van der Schueren, E., Dewit, L. and Dixon, D.O. Direct estimation of latent time for radiation injury in late-responding normal tissues: gut, lung, and spinal cord. Int. J. Radiat. Biol. 55: 27-43, 1989. 7 Bentzen, S.M., Thames, H . D . , Tucker, S.L. a n d Smith, C. New options in direct analysis of dose-response data. Int. J. Radiat. Biol. 57: 221-225, 1990. 8 Carpenter, S. G. and Raju, M . R . Residual damage in the mouse
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foot after exposure to heavy particles. Radiology 138: 483-485, 1981. Chen, F. D. and Hendry, J . H . Re-irradiation of mouse skin: similarity of dose reduction for healing and macrocolony endpoints. Radiother. Oncol. 11: 153-159, 1988. Guttenberger, R. and Thames, H . D . Kinetics of recovery from sublethal radiation damage. Br. J. Radiol. 62:1108-1109, 1989. Hopewell, J. W. and Van den Aardweg, G. M . J . Current concepts of dose fractionation in radiotherapy. Normal tissue tolerance. Br. J. Radiol. Suppl. 22: 88-93, 1988. Hornsey, S., Myers, R. and Warren, P. Residual injury in the spinal cord after treatment with x rays or neutrons. Br. J. Radiol. 55: 516-519, 1982. Knowles, J . F . The radiosensitivity of the guinea-pig spinal cord to x-rays: the effect of retreatment at one year and the effect of age at the time of irradiation. Int. J. Radiat. Biol. 44: 433-442, 1983. Litchfield, J. T. and Wilcoxon, F. A simplified method of evaluating dose-effect experiments. J. Pharm. Exp. Ther. 96: 99-113, 1949. Mould, R . F . Calculation of survival rates by the life table and other methods. Clin. Radiol. 27: 33-38, 1976. Moulder, J. E. and Fish, B.L. Repair of sublethal radiation injury in the rat kidney. In: Radiation Research. A TwentiethCentury Perspective. pp. 238 (Abstract). Editors: J . D . Chapman, W. C. Dewey and G. F. Whitmore. Academic Press, Inc., Toronto, 1991. Ruifrok, A. C. C., Kleiboer, B. and van der Kogel, A.J. Re-
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irradiation tolerance of the immature rat spinal cord. Radiother. Oncol. 23: 249-256, 1992. Stewart, F. A., Luts, A. and Lebesque, J.V. The lack of longterm recovery and reirradiation tolerance in the mouse kidney. Int. J. Radiat. Biol. 56: 449-462, 1989. Stewart, F. A. and Oussoren, Y. Re-irradiation of mouse kidneys: a comparison of re-treatment tolerance after single and fractionated partial tolerance doses. Int. J. Radiat. Biol. 58: 531544, 1990. Stewart, F. A., Oussoren, Y. and Luts, A. Long-term recovery and reirradiation tolerance of mouse bladder. Int. J. Radiat. Oncol. Biol. Phys. 18: 1399-1406, 1990. Terry, N. H. A., Tucker, S. L. and Travis, E.L. Residual damage in murine lung assessed by pneumonitis. Int. J. Radiat. Oncol. Biol. Phys. 14: 929-938, 1988. Terry, N. H. A., Tucker, S. L. and Travis, E.L. Time course of loss of residual radiation damage in murine skin assessed by retreatment. Int. J. Radiat. Biol. 55: 271-283, 1989. Thames, H . D . An "incomplete-repair" model for survival after fractionated and continuous irradiations. Int. J. Radiat. Biol. 47: 319-339, 1985. Thames, H . D . Repair kinetics in tissues: alternative models. Radiother. Oncol. 14: 321-327, 1989. Thames, H. D., Ang, K. K., Stewart, F. A. and van der Schueren, E. Does incomplete repair explain the apparent failure of the basic LQ model to predict spinal cord and kidney responses to low doses per fraction? Int. J. Radiat. Biol. 54: 13-19,1988. Thames, H. D., Rozell, M. E., Tucker, S. L., Ang, K. K. Fisher, D . R . and Travis, E.L. Direct analysis of quantal response data. Int. J. Radiat. Biol. 49: 999-1009, 1986. Thames, H . D . and Hendry, J . H . Fractionation in Radio-
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therapy. Taylor and Francis, London, New York, Philadelphia, 1987. Turesson, I. and Thames, H . D . Repair capacity and kinetics of human skin during fractionated radiotherapy: erythema, desquamation, and telangiectasia after 3 and 5 year's follow-up. Radiother. Oncol. 15: 169-188, 1989. Van den Aardweg, G. J. M. J. and Hopewell, J . W . The kinetics of repair for sublethal radiation-induced damage in the pig epidermis: an interpretation based on a fast and a slow component of repair. Radiother. Oncol. 23: 94-104, 1992. Van den Aardweg, G. J. M. J., Hopewell, J. W. and Simmonds, R . H . Repair and recovery in the epithelial and vascular connective tissues of pig skin after irradiation. Radiother. Oncol. 11: 73-82, 1988. Van der Kogel, A.J. Late effects of radiation on the spinal cord. Dose-effect relationships and pathogenesis. Monograph, publication of the Radiobiological Institute TNO, Rijswijk, The Netherlands, 1979. Van der Kogel, A.J. The nervous system: Radiobiology and experimental pathology. In: Medical Radiology. Radiopathology of organs and tissues, pp. 191-212. Editors: E. Scherer, Ch. Streffer and K.-R. Trott. Springer-Verlag, Berlin, Heidelberg, 1991. Van der Kogel, A. J., Sissingh, H. A. and Zoetelief, J. Effect of x rays and neutrons on the repair and regeneration in the rat spinal cord. Int. J. Radiat. Oncol. Biol. Phys. 8: 2095-2097, 1982. Van Rongen, E. Recovery from radiation damage in mouse lungs: interpretation in terms of two rates of repair (Abstract). Int. J. Radiat. Biol. 59: 1283, 1991. White, A. and Hornsey, S. Time dependent repair of radiation damage in the rat spinal cord after x-rays and neutrons. Eur. J. Cancer 16: 957-962, 1980.
295
RADION 01028
Fractionation sensitivity of the rat cervical spinal cord during radiation retreatment A. C. C. Ruifrok, B. J. Kleiboer and A. J. v a n der Kogel Institute of Radiotherapy, University of Nijmegen, Nijmegen, The Netherlands (Received 4 February 1992, revision received 7 April 1992, accepted 21 April 1992)
Key words: Spinal cord; Radiation myelopathy, Long-term recovery; Retreatment, Fractionation; Late effect
Summary Data concerning the fractionation sensitivity of normal tissues during radiation retreatment are limited. Experiments were performed to investigate whether the fractionation sensitivity of the rat cervical spinal cord is changed during retreatment 6 months after a first dose of 15 Gy, representing about half the biologically effective dose for induction of paresis. After a 6 months interval, the long-term recovery from the first treatment was about 45 %. The fractionation sensitivity of the rat cervical spinal cord during reirradiation was not significantly different from the fractionation sensitivity of not previously irradiated control rats, with an ~/flratio of 2.3 Gy in control rats and 1.9 Gy during reirradiation of the spinal cord. An additional observation from these experiments was the presence of incomplete repair after fractionated treatment with 2 fractions of 3 Gy per day with 10-h intervals.
Introduction For several acute as well as late reacting normal tissues, animal studies concerning retreatment tolerance have been performed. Most of the work concerning longterm recovery has been directed to the time course and extent of recovery after single dose or fractionated first treatments and single dose test treatments [8,9,12,13,17,19-22,30,33,35]. Data concerning the fractionation sensitivity during retreatment are limited. Stewart et al. [ 18] showed that in mouse kidney there was no reduction in fractionation sensitivity when fractionated irradiation was given to mice irradiated 26 weeks before. However, Turesson and Thames [28] reported an increase in ~/fl ratio as a function of overall treatment time during treatments of more than 4 weeks for the early reactions of erythema and desquamation of human skin, suggesting a decreased fractionation sensitivity, while no clear change in ~/fl for the late reaction of telanglectasia was found [28]. Although prolonged treatments are not directly comparable to
reirradiation, the data indicate that in some situations the fractionation sensitivity of a tissue may change depending on previous radiation treatments. This may have important consequences for the normal tissue tolerance and the optimal fractionation schemes for retreatment in case of tumor recurrences in previously irradiated sites. The present experiments were performed to investigate whether the fractionation sensitivity of the rat spinal cord is changed during radiation retreatment.
Methods and materials Female Wistar rats ( C P B / W U ) were used in this study at an age of 14-20 weeks (adult rats). The rats were housed in macrolon cages and provided with water and food ad libitum. Prior to irradiation, the animals were anesthetized with Ethrane inhalation [5]. Positioning was facilitated using a lucite fixation setup, enabling us to irradiate six animals simultaneously. Irradiations were performed on a linear accelerator
Address for correspondence: A. J. van der Kogel, Institute of Radiotherapy, University of Nijraegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.
296 at a focus-skin distance of 100 cm. Exposure of a 18 mm segment of the spinal cord (C1 through T1-T2) was carried out with 4 MeV photons at a dose rate of 2.2 Gy/min. The head and body were shielded with 70 mm lead blocks close to the skin. All rats received a first irradiation dose of 15 Gy. A second course of irradiation was given either at one day (control), or at 6 months after this first dose, with "single dose" or fractionated treatments, with fraction sizes of 3, 4 and 6 Gy. The 4 and 6 Gy fraction treatments were given 5 days per week; the 3 Gy per fraction treatments were given 5 days per week with 2 fractions per day with a 10-h interval. Also a control treatment with one fraction of 3 Gy per day was performed. Each experiment comprised 5-6 dose levels with 5-6 animals per dose group. Examinations for signs of neurological impairment were performed 3 times a week, starting 3 months after the last treatment course. Animals were scored as responders when they showed regular dragging of their feet with palmar flexion, dragging of extended forelegs, or inability to walk on their forelegs when lifted by the tail. When these definite signs of paresis were seen, rats were sacrificed. Follow-up of the animals was continued up to 300 days after the last irradiation. The sacrificed animals were perfused with saline and 4~o buffered formaldehyde solution with a standard infusion set at a pressure of 100-120 cm H20. The irradiated part of the spinal column was dissected out for histological examination. The spinal columns were decalcified in 20~o formic acid, 4 ~ sodium formate solution, and embedded in paraffin. Haematoxylineosin stained sections of 6 #m thickness were routinely examined for histological damage. Rats of which the neurological symptoms could be attributed to spinal cord compression by a tumor, or damage to vertebrae resulting in spinal cord compression, were not recorded as responders but as intercurrent deaths. Response rates were corrected with the life-table method when intercurrent deaths occurred [1,15]. Dose-response curves were constructed by probitanalysis. Significance of differences in doses resulting in 50~o probability of paresis (50~o effective dose, EDso) were tested using the Z2-test [ 14]. Fitting of the linear quadratic (LQ) model to the data was done with the direct data analysis method of Thames et al. [26] using a computer program provided by the Department of Biomathematics in Houston [7]. Results
Irradiation of the cervical spinal cord of the rats resulted in paresis of the forelegs after latent periods of
TABLE I Latent times for development of paresis after a first dose of 15 Gy, followed by fractionated irradiation after one day or 6 months, at an isoeffective range of EDso-ED99. Treatment
Latency after first fraction* (days) mean _+SD (median)
15 G y + S D 15 Gy + 6 Gy/fx 15 G y + 4 Gy/fx 15 G y + 3 Gy/fx; 2 fx/day
Interval one day
Interval 6 months
233+18(235) 187 + 14 (192) 199+25 (196) 218_+25 (221)
160+21(162) 152 + 23 (150) 156_+25 (146) 165+60(151)
* Overall treatment time for fractionated treatments with a second course after one day was 8-18 days, for fractionated retreatments after 6 months 11-29 days.
5 to 7 months. In Table I, the latent periods after treatments associated with 80-99~o probability of paresis (EDso-ED99), are summarized. Following control treatments the latency tends to increase with decreasing fraction size, however, this increase is not significant due to the large spread in latency times after the treatments. Second course irradiation with a single dose even results in a longer latent time than observed after treatment with the largest fraction size of 6 Gy. Following irradiation 6 months after the first dose all latent times are shortened by about 1-2 months compared to the latency after control treatments. No indication of fraction size dependent latency is observed after a 6 months interval. Figure 1 shows the probit curves for development of paresis during the follow-up period of 300 days after
% paretic
loo%
•
,t
80%"
/
60%
!
m
•
40% 20%" 0%
, 0
20
40
610
8Uo
1 O0
D o s e (Gy)
Fig. 1. Dose-response curves for paresis after tionated control treatments one day after a --.--, SD; ---------I~, 6 Gy/fx; - - & - - , 3 Gy/fx, 1 fx/day; .... [] .... ,3 Gy/fx, 2 fx/day. fidence interval.
single dose and fracfirst dose of 15 Gy. 4 Gy/fx; - - U - - , Error bars: 95~ con-
297 "single dose" and fractionated control treatments. The solid lines represent "single dose" and fractionated treatments given with time intervals of at least 24 h; the dotted line represents the result of fractionated treatment with 2 fractions of 3 Gy per day with a time interval of 10 h. The EDso values calculated from these probit curves are summarized in Table II. As can be seen, the EDso increases with decreasing fraction size when the time interval between fractions was at least 24 h. Treatment with 2 fractions of 3 Gy per day resuited in a significantly lower EDso of 42.0 Gy compared to the EDso of 56.6 Gy after treatment with 1 fraction per day (p < 0.05). In Fig. 2, the probit curves for single dose and fractionated reirradiation 6 months after the first dose of 15 Gy are shown. The solid lines represent the single dose and one fraction per day treatments. The dotted line represents the treatment with 2 fractions of 3 Gy per day with an interval time of 10 h. The EDso values
TABLE II EDso values for paresis after cervical spnal cord irradiation with a first dose of 15 Gy, followed by a second irradiation course after one day or 6 months. Treatment
15 15 15 15 15
EDso (Gy) of the second irradiation course (95 % confidence interval)
Gy + SD Gy + 6 Gy/fx Gy + 4 Gy/fx G y + 3 Gy/fx; 1 fx/day Gy + 3 Gy/fx; 2 fx/day
Interval one day
Interval 6 months
16.2 (15.5-17.3) 36.8 (34.2-40.4) 47.2 (44.4-49.9) 56.6 (50.2-98.4) 42.0 (35.2-49.3)
18.5 (17.5-19.7) 43.2 (30.7-50.1) 65.1 (57.1-75.0) 54.9 (45.4-60.9)
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610 (Gy)
Function paramter values of direct data analysis of rats irradiated one day and 6 months after 15 Gy priming irradiation treatment. Parameter
InK 10.~ (Gy- 1) 100.fl(Gy -2) ~/fl(Gy)
Function parameters (95% confidence interval) One day interval
6 months interval
5.8 0.47 2,1 2.3
5.2 0.29 1.5 1.9
(4.5-7.2) (0.32-0.62) (1.5-2.6) (1.5-3.3)
(3.4-7.0) (0.13-0.46) (1.0-2.1) (0.7-3.8)
Function: E = - InK + otD + ~D21N, with E = - log survival for isoeffect, K the number of "tissue rescuing units", ~ and fl the parameters of the LQ model, D the total dose and N the number of fractions [26].
calculated from these curves are summarized in Table II. Also after reirradiation 6 months after the first dose of 15 Gy the EDso increases with decreasing fraction size when daily fractions were given; however, treatment with 2 fractions of 3 Gy per day resulted in an ED50 of about 55 Gy, which is considerably lower than the ED50 of about 75 Gy expected on the basis of the LQ model with complete repair between the fractions. To estimate the fractionation sensitivity of control rats and rats treated 6 months after the first dose of 15 Gy, the paresis data were analyzed using the direct analysis method of Thames et al. [26]. Because the results of the experiments with 2 fractions of 3 Gy per day were different from the predictions on the basis of the LQ model, the calculations were performed using the data of "single dose" and daily fractionation treatments only. The results of these calculations are summarized in Table III. Although the results suggest a minor decrease in the absolute values of ct as well as during the reirradiation treatment" after 6 months, we observed no significant difference in the ct/pratio for irradiation at one day or 6 months after a first dose of 15 Gy; the ct//~ratio is about 2 Gy after second course irradiation after one day as well as during reirradiation after 6 months.
•
Discussion
, , 40 Dose
TABLE III
810
1 00
Fig. 2. Dose-response curves for paresis after single dose and fractionated second course irradiations 6 months after a first dose of 15 Gy. - - e - - , SD; - - ~ I ~ - - , 6 Gy/fx; - - & - - , 4 Gy/fx; .... [] .... ,3 Gy/fx, 2 fx/day. Error bars: 95% confidence interval.
The latency to paresis after fractionated irradiation treatment of the spinal cord has been suggested to be fraction-size dependent [6]. Although the latency data of the experiments with a second irradiation course at one day after a single dose of 15 Gy also suggest a fraction size dependent latency, the differences in the latent times are not significant (p > 0.05) (Table I). The
298 latent times after a second irradiation course after 6 months give no suggestion of fraction size dependence at all. Therefore, the data were analyzed using direct estimation of the fractionation characteristics without latent time analysis [6,26]. The experiments presented in this paper were designed to determine the fractionation sensitivity of the rat cervical spinal cord during retreatment. Because of the expected high tolerance dose for a retreatment using 3 Gy fractions, this particular schedule was carried out with 2 fractions per day, to limit the overall time to about 4 weeks for the highest dose. The chosen interval time of 10 h between the 2 fractions given on one day was assumed to be sufficient for complete SLD repair according to reported repair half-times (ill2) of 1.4 to 1.9 h for rat spinal cord [3,4,25]. However, as shown in Fig. 2 and Table II, the 3 Gy fractionation resulted in a lower EDso than expected; the observed ED~o was even lower than after treatments with 1 fraction of 4 Gy/day. This indicated that a 10-h interval was not sufficient for complete repair in this experiment. To investigate this apparent loss of tolerance with 2 fractions per day, control treatments were performed with 1 and 2 fractions of 3 Gy/day. These latter experiments supported the notion of incomplete repair for 2 fractions per day in the retreatment experiments; the 2 fractions per day treatments resulted in a significantly ( p < 0.05) lower EDso than the single fraction per day treatment. Assuming that the difference in EDso values after 1 and 2 fractions per day is the result of incomplete repair, these data indicate a t,/: of about 5.5 h when mono-exponential decay of SLD is assumed [23]. More recently, it has been suggested that repair in the spinal cord may exhibit a two-component decay of the SLD. Hopewell and van den Aardweg [11] suggested t,/~ values of 10.6 min and 2.4 h for spinal cord on the basis of graphical analysis of the data of Ang et al. [4]. Although this method of analysis has been criticized [ 10], there is a trend in the presented data suggesting that the assumption of mono-exponential decay of SLD is too simple. Ang et al. [2] reported for the rat spinal cord t,/2 values of 0.7 and 4 h, with 6 5 ~ of the damage repaired by the long repair tl/2, when experimental data were fitted by a bi-exponential repair model. Also for the rat kidney [16] and lung [34], and pig skin [29] indications of two-component repair of SLD have been reported. The decay of SLD may be dose-dependent [4,16], show repair saturation [24], may be interval-dependent [10], or may be a two-component process [2,11,16,22,29,34]. However, as long as no sufficient
data are available to determine the exact nature of SLD repair, mono-exponential repair models usually provide an adequate description of normal tissue responses to fractionated irradiation. The present experiments were not designed to determine the kinetics of SLD repair, and are insufficient to determine repair times in a twocomponent model; nevertheless, the data suggested the presence of a long repair time, and clearly show a significant continued recovery between 10 and 24 h. Retreatment of previously irradiated cervical spinal cord after 6 months shows that the tolerance of the spinal cord is increased compared to treatment directly after the first dose. When expressed as a percentage of the total effect (TE) according to the LQ model [ 17,27], the first dose of 15 Gy represents about 45 ~ of the TE, with a remaining tolerance of 55~o TE. The reirradiation tolerance for single dose and fractionated retreatments after 6 months recovery represents about 7 5 ~ TE, indicating that the total dose that can be given in two treatment courses with 6 months interval represents about 120~o of the TE that can be given in a single treatment course. This value is similar to the long-term recovery found previously for immature rats of the same strain [17], but somewhat lower than reported before for adult Wag/Rij rats [33]. The data indicate that only about 45 ~o of the damage of the first treatment is recovered at 6 months. Also the shortening of the latent time after irradiation at 6 months compared to one day is in accordance with considerable damage from the first treatment still being present after 6 months. Because the data obtained for 2 fractions per day were clearly influenced by incomplete repair, only the 1 fraction per day was used for analysis of fractionation sensitivity directly after the first dose and during retreatment after 6 months. The results of this analysis are presented in Table III. The absolute values of the parameters ~ and fl of the LQ model seem to be slightly different for control treatments and reirradiation after 6 months; however, these values are strongly influenced by experimental variation in the original data, and the confidence intervals show that the differences are not significant. The most important result of the direct analysis is that the calculated ~/flratio during control treatment and reirradiation 6 months after 15 Gy are not significantly different, 2.3 Gy during control treatment and 1.9 Gy during a reirradiation treatment after 6 months, indicating that the capacity of SLD repair is not changed during retreatment. This means that for the rat spinal cord, like for the mouse kidney [ 17], there is no indication for reduction in capacity of SLD repair during reirradiation. The 2 fraction per day data suggest
299 that also the rate of repair in the rat spinal cord is comparably slow during control treatment and reirradiation after 6 months. The present data of the cervical spinal cord and those of the mouse kidney [ 18] show no change in the fractionation sensitivity during reirradiation. However, Turesson and Thames [28] found an increase in ~/fl for the early reactions of erythema and desquamation of human skin during prolonged treatment for more than 4 weeks, while no clear change in ~/fl for the late reaction of telangiectasia was found. These present and previously reported [ 18,28] results may indicate that in late reacting tissues, with normally a low cellular turnover, cellular kinetics may be not or only slightly changed during prolonged irradiation or reirradiation, while in acute responding tissues redistribution in the cell cycle and proliferative response may cause a change in the fractionation sensitivity. An acute proliferative response may result in a greater heterogeneity in the sensitivity of the target cells during prolonged treatment or reirradiation, resulting in a change in the ~/flvalue determined. A possible change in the fractionation sensitivity may also depend on the timing of reirradiation; the air ratio may be changed at the moment of maxi-
mal cellular responses to the first irradiation, while no significant change in ~/fl may be found before or after the maximal response. Summarizing, the present experiments indicate that: (1)the fractionation sensitivity of the cervical spinal cord during retreatment at 6 months after a first dose of about 45 ~o TE is not significantly changed compared to the fractionation sensitivity of the cervical spinal cord of control animals; (2) at 6 months after a first dose to the cervical spinal cord, representing 45 ~ TE, about 45 ~o the damage of the first dose has been recovered; and (3) in the rat cervical spinal cord significant repair continues to take place between 10 and 24 h.
Acknowledgements The author wish to thank W. F. M. Brouwer for physics support, T. Oostendorp for expert technical assistance, J. Koedam and coworkers of the Central Animal Laboratory for excellent collaboration, and H . D . Thames and C. Smith for providing the computer program for direct data analysis. This work was supported by grant N U K C 88-2 from the Dutch Cancer Society.
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