ht. J. Radiamn Oncology Bid Phys.. Vol. 24. pp. W-5 Printed in the U.S.A. All rights reserved.
0364J-3016/92 $5.00 + .oO Copyri@t Q 1992 Pergamon Press Ltd
IO
??Biology Original Contribution
RADIATION TOLERANCE AND FRACTIONATION SENSITIVITY OF THE DEVELOPING RAT CERVICAL SPINAL CORD ARNOUT
C. C. RUIFROK, PH.D., BERT J. KLEIBOER, AND ALBERT J. VAN DER KOGEL, PH.D.
Institute of Radiotherapy,
B.Sc.
University of Nijmegen, The Netherlands
To investigate the influence of age at irradiation on single dose radiation tolerance and fractionation sensitivity, the cervical spinal cord of rats was irradiated at the age of 1 week and at 15-18 weeks (adult). While the main histological lesions seem to be comparable after irradiation at the two ages, differences were found in single dose tolerance, latency to paresis due to white matter lesions, and fractionation sensitivity. The 50% effect dose (EDso) for single dose irradiation at one week was 19.5 Gy, which is only lo%, but significantly (p < 0.05), lower than the EDg of about 21.5 Gy at 3 weeks and above. The latency to paresis was clearly influenced by the age at irradiation. The latency in the rats irradiated at 1 week was about 2 weeks, while for adult rats a latency of about 8 months was observed. The fractionation sensitivity for irradiation at 1 week was lower than the fractionation sensitivity of the adult rats; the (Y/B value at 1 week was estimated to be 4.5 Gy, while for the adult rats an (Y/B value of 1.8 Gy was found. As a consequence, the observed small difference in tolerance to single doses between 1 week-old and adult rats is further enhanced after fractionated irradiation. During prolonged irradiation treatments this decreased tolerance may be compensated by a higher proliferation rate in the immature central nervous system. The results of the present experiments indicate that, for a single tissue and endpoint, paresis due to white matter lesions in the rat cervical spinal cord, the latency to expression of damage and the fractionation sensitivity clearly change with age at irradiation. Age, Spinal cord, Radiation tolerance, Radiation myelopathy,
Many developing tissues are more vulnerable to the effects of radiation than tissues of adult animals, presumably because of the more active proliferation. Age dependent differences in radiation response have been described for rat skin (9) mouse kidney (4), pig kidney (16), and guineapig spinal cord (11). However, very little is known about the fractionation sensitivity of the developing tissues. Because it may be expected that not only tolerance, but also fractionation sensitivity is decreased in actively proliferating tissues, we investigated single dose tolerance and fractionation sensitivity of the cervical spinal cord of 7 day-old and adult rats.
AND
Late effects, Development.
strain at 15- 18 weeks (adult rats). The rats were housed in macrolon cages and provided with water and food ad libitum. The 7 day-old rats were housed 10 in a cage with one adult foster-mother until weaning at the age of 3 weeks.
INTRODUCTION
METHODS
Fractionation,
Irradiations Prior to irradiation, the animals were anesthetized with Ethrane inhalation (2). Positioning was facilitated using a lucite fixation setup, enabling us to irradiate six animals simultaneously. All irradiations were performed with 4 MV photons on a linear accelerator at a focus-spinal cord distance of 100 cm. Dosimetry was performed at the level of the spinal cord, with appropriate sized phantom measurements as well as TLD measurements in situ in killed animals. In the 1 week-old rats exposure of a 8 mm segment of the cervical and upper thoracic spinal cord (Cl through T4-T5) was carried out at a dose-rate of 2.1 Gy/ min. The adult rats were irradiated on a 18 mm cervical
MATERIALS
Animals Male and female Wistar rats (CPB/WU) were used in this study at an age of 7 days and female rats of the same
Reprint requests to: A. J. van der Kogel, Ph.D., Institute of Radiotherapy, University of Nijmegen, P.O. Box 9 101, 6500 HB Nijmegen, The Netherlands. Acknowledgements-The authors wish to thank W. F. M. Brouwer for physics support, T. Oostendorp for expert technical as-
sistance and J. Koedam and coworkers of the Central Animal Laboratory for excellent collaboration. This work was supported by grant NUKC 88-2 from the Dutch Cancer Society. Accepted for publication 2 1 April 1992. 505
506
I. J. Radiation Oncology 0 Biology 0 Physics
segment (Cl through Tl -T2) at a dose-rate of 2.2 Gy/ min. The head and body of the rats were shielded with 70 mm lead close to the skin. Experimental set-up Each experiment comprised 5-6 dose levels with 5-6 animals per dose group, with the 7 day-old male and female animals distributed equally over the dose groups. Examinations for signs of neurological impairment were performed daily on the rats irradiated at 7 days and 3 times a week on the adult rats. Animals were scored as responders when they showed regular dragging of their feet with palmar flexion, dragging of extended forelegs, clr inah;lit.r tn w~llr nn th&r fr\rs&-nr wh.an l;ftr.& h., the “I IUGe”,l‘LJ C” ..UIR “IA Cllrll nvnrnvgu “.ll\rll I‘ICbU “J U,b tail. When these definite signs of paresis or paralysis were seen, rats were killed. Follow-up of the animals was continued up to 200 days after the last irradiation in rats that received their first irradiation at 7 days, and 300 days after the last irradiation in adult rats. The killed animals were perfused with saline and 4% buffered formaldehyde solution with a standard infusion set at a pressure of lOO- 120 cm H20. The irradiated part of the spinal column was dissected out for histological examination. The spinal columns were decalcified in 20% formic acid, 4% sodium formate solution, and embedded in paraffin. Haematoxylin eosin stained sections of 6 pm thickness were routinely examined for histological damage. During the follow-up period occasionally rats were lost because of respiratory problems (2%), tumor development (1%) or unknown causes (8%); these rats were scored as intercurrent deaths. When intercurrent deaths occurred, response rates were corrected with the life-table method (1, 14). Dose-response curves were constructed by probitanalysis. When no groups with a response between 0 and 100% were observed, probit curves were plotted by eye, and the highest dose giving no response and the lowest dose giving 100% response were considered to represent the 95% confidence interval__ (29). nfdi&r\__ ,_ Significance _.~___________ ences in doses resulting in 50% probability of paresis (50% effective dose, EDSo) were tested using the X2 test (12). Fitting of the linear quadratic (LQ) model was done using the direct data analysis method of Thames et al. (2 1).
Volume 24, Number 3, I992
rats paresis of the forelegs developed 150-270 days after single dose treatments of 22 to 28 Gy, with the shorter latencies after the higher doses. In 1 week-old as well as adult animals small focal areas of necrosis and diffuse demyelination were the most prominent histological lesions. A more detailed analysis of histological damage in the cervical spinal cord, after irradiation at different ages, will be the subject of a separate paper. Figure 1 shows the probit curves for paresis of the forelegs after single-dose irradiation in the 1 week-old and adult rats. A small but significant (p < 0.05) difference in response after singledose irradiation of 1 week-old and adult rats is observed; the EDso value for 1 week-old rats is 19.5 Gy (18.7-20.3 r.,,. lva~ cn-_ -f qrl..it rq+cl -f 3 1 n UJ) a
0364J-3016/92 $5.00 + .oO Copyri@t Q 1992 Pergamon Press Ltd
IO
??Biology Original Contribution
RADIATION TOLERANCE AND FRACTIONATION SENSITIVITY OF THE DEVELOPING RAT CERVICAL SPINAL CORD ARNOUT
C. C. RUIFROK, PH.D., BERT J. KLEIBOER, AND ALBERT J. VAN DER KOGEL, PH.D.
Institute of Radiotherapy,
B.Sc.
University of Nijmegen, The Netherlands
To investigate the influence of age at irradiation on single dose radiation tolerance and fractionation sensitivity, the cervical spinal cord of rats was irradiated at the age of 1 week and at 15-18 weeks (adult). While the main histological lesions seem to be comparable after irradiation at the two ages, differences were found in single dose tolerance, latency to paresis due to white matter lesions, and fractionation sensitivity. The 50% effect dose (EDso) for single dose irradiation at one week was 19.5 Gy, which is only lo%, but significantly (p < 0.05), lower than the EDg of about 21.5 Gy at 3 weeks and above. The latency to paresis was clearly influenced by the age at irradiation. The latency in the rats irradiated at 1 week was about 2 weeks, while for adult rats a latency of about 8 months was observed. The fractionation sensitivity for irradiation at 1 week was lower than the fractionation sensitivity of the adult rats; the (Y/B value at 1 week was estimated to be 4.5 Gy, while for the adult rats an (Y/B value of 1.8 Gy was found. As a consequence, the observed small difference in tolerance to single doses between 1 week-old and adult rats is further enhanced after fractionated irradiation. During prolonged irradiation treatments this decreased tolerance may be compensated by a higher proliferation rate in the immature central nervous system. The results of the present experiments indicate that, for a single tissue and endpoint, paresis due to white matter lesions in the rat cervical spinal cord, the latency to expression of damage and the fractionation sensitivity clearly change with age at irradiation. Age, Spinal cord, Radiation tolerance, Radiation myelopathy,
Many developing tissues are more vulnerable to the effects of radiation than tissues of adult animals, presumably because of the more active proliferation. Age dependent differences in radiation response have been described for rat skin (9) mouse kidney (4), pig kidney (16), and guineapig spinal cord (11). However, very little is known about the fractionation sensitivity of the developing tissues. Because it may be expected that not only tolerance, but also fractionation sensitivity is decreased in actively proliferating tissues, we investigated single dose tolerance and fractionation sensitivity of the cervical spinal cord of 7 day-old and adult rats.
AND
Late effects, Development.
strain at 15- 18 weeks (adult rats). The rats were housed in macrolon cages and provided with water and food ad libitum. The 7 day-old rats were housed 10 in a cage with one adult foster-mother until weaning at the age of 3 weeks.
INTRODUCTION
METHODS
Fractionation,
Irradiations Prior to irradiation, the animals were anesthetized with Ethrane inhalation (2). Positioning was facilitated using a lucite fixation setup, enabling us to irradiate six animals simultaneously. All irradiations were performed with 4 MV photons on a linear accelerator at a focus-spinal cord distance of 100 cm. Dosimetry was performed at the level of the spinal cord, with appropriate sized phantom measurements as well as TLD measurements in situ in killed animals. In the 1 week-old rats exposure of a 8 mm segment of the cervical and upper thoracic spinal cord (Cl through T4-T5) was carried out at a dose-rate of 2.1 Gy/ min. The adult rats were irradiated on a 18 mm cervical
MATERIALS
Animals Male and female Wistar rats (CPB/WU) were used in this study at an age of 7 days and female rats of the same
Reprint requests to: A. J. van der Kogel, Ph.D., Institute of Radiotherapy, University of Nijmegen, P.O. Box 9 101, 6500 HB Nijmegen, The Netherlands. Acknowledgements-The authors wish to thank W. F. M. Brouwer for physics support, T. Oostendorp for expert technical as-
sistance and J. Koedam and coworkers of the Central Animal Laboratory for excellent collaboration. This work was supported by grant NUKC 88-2 from the Dutch Cancer Society. Accepted for publication 2 1 April 1992. 505
506
I. J. Radiation Oncology 0 Biology 0 Physics
segment (Cl through Tl -T2) at a dose-rate of 2.2 Gy/ min. The head and body of the rats were shielded with 70 mm lead close to the skin. Experimental set-up Each experiment comprised 5-6 dose levels with 5-6 animals per dose group, with the 7 day-old male and female animals distributed equally over the dose groups. Examinations for signs of neurological impairment were performed daily on the rats irradiated at 7 days and 3 times a week on the adult rats. Animals were scored as responders when they showed regular dragging of their feet with palmar flexion, dragging of extended forelegs, clr inah;lit.r tn w~llr nn th&r fr\rs&-nr wh.an l;ftr.& h., the “I IUGe”,l‘LJ C” ..UIR “IA Cllrll nvnrnvgu “.ll\rll I‘ICbU “J U,b tail. When these definite signs of paresis or paralysis were seen, rats were killed. Follow-up of the animals was continued up to 200 days after the last irradiation in rats that received their first irradiation at 7 days, and 300 days after the last irradiation in adult rats. The killed animals were perfused with saline and 4% buffered formaldehyde solution with a standard infusion set at a pressure of lOO- 120 cm H20. The irradiated part of the spinal column was dissected out for histological examination. The spinal columns were decalcified in 20% formic acid, 4% sodium formate solution, and embedded in paraffin. Haematoxylin eosin stained sections of 6 pm thickness were routinely examined for histological damage. During the follow-up period occasionally rats were lost because of respiratory problems (2%), tumor development (1%) or unknown causes (8%); these rats were scored as intercurrent deaths. When intercurrent deaths occurred, response rates were corrected with the life-table method (1, 14). Dose-response curves were constructed by probitanalysis. When no groups with a response between 0 and 100% were observed, probit curves were plotted by eye, and the highest dose giving no response and the lowest dose giving 100% response were considered to represent the 95% confidence interval__ (29). nfdi&r\__ ,_ Significance _.~___________ ences in doses resulting in 50% probability of paresis (50% effective dose, EDSo) were tested using the X2 test (12). Fitting of the linear quadratic (LQ) model was done using the direct data analysis method of Thames et al. (2 1).
Volume 24, Number 3, I992
rats paresis of the forelegs developed 150-270 days after single dose treatments of 22 to 28 Gy, with the shorter latencies after the higher doses. In 1 week-old as well as adult animals small focal areas of necrosis and diffuse demyelination were the most prominent histological lesions. A more detailed analysis of histological damage in the cervical spinal cord, after irradiation at different ages, will be the subject of a separate paper. Figure 1 shows the probit curves for paresis of the forelegs after single-dose irradiation in the 1 week-old and adult rats. A small but significant (p < 0.05) difference in response after singledose irradiation of 1 week-old and adult rats is observed; the EDso value for 1 week-old rats is 19.5 Gy (18.7-20.3 r.,,. lva~ cn-_ -f qrl..it rq+cl -f 3 1 n UJ) a
