Abstract
The central nervous system (CNS) is among the critical late-responding normal tissues which influence radiation treatment planning and dose prescription. Both mouse and rat spinal cord are established radiobiological models. As late reactions in rodents develop many months after irradiation, long-term follow-up is necessary. Development of radioprotective strategies and refinement of optimum dose and administration schedules are therefore quite complicated and time consuming. Obviously, surrogate endpoints that allow for screening of radioprotective agents and optimisation of dosing with shorter follow-up would speed up and facilitate this type of radiobiological research. Such surrogate endpoints should be analyzable during the clinically asymptomatic latent period, ideally as early as possible. As unrepaired cellular damage is a prerequisite for overt side-effects of radiotherapy, detection of residual damage might predict for radiation myelopathy. Immunohistochemical evaluation of irradiated mouse spinal cord with phosphorylated histone H2AX (γ-H2AX) was performed. Preliminary data suggest that residual damage can be detected in mouse spinal cord and that such foci continue to be detectable over a long time period, which clearly extends into the phase where development of late reactions starts. Our initial findings justify further research which attempts to correlate γ-H2AX foci with the primary endpoint, i.e. radiation myelopathy. It can then be explored whether a given radioprotective agent reduces the number of foci and whether this translates into reduced incidence of radiation myelopathy. A rapid and reliable animal model would allow for screening of a large number of candidate drugs that might modify the radiation response of the spinal cord.
When treating cancer patients with ionising radiation, one has to achieve a favourable balance between tumour control and the probability of normal tissue complications. Recent developments, such as intensity-modulated radiotherapy, have improved the ability to better shape the dose distribution to the target volume and thus spare more of nearby critical organs. Despite these advances, development of strategies of response modification, in this case protection of healthy tissue, would further improve the therapeutic ratio. The central nervous system (CNS) is among the critical late-responding normal tissues which influence radiation treatment planning and dose prescription (1-3). Several groups have studied experimental CNS radiotherapy in rodents, and both mouse and rat spinal cord are among the established models (4-12). As late reactions in rodents develop many months after irradiation, long-term follow-up is necessary. Development of radioprotective strategies and refinement of optimum dose and administration schedules are therefore quite complicated and time consuming (5-7). Obviously, surrogate endpoints allowing for screening of radioprotective agents and optimisation of dosing with shorter follow-up would speed up and facilitate this type of radiobiological research.
Ionising radiation causes DNA damage in cells and tissues, which if left unrepaired might cause clinically apparent side-effects in healthy organs. DNA damage can be assayed by the formation and resolution of nuclear DNA repair foci (13, 14). These foci, e.g. phosphorylated histone H2AX (γ-H2AX), can be detected immunohistochemically in irradiated tissue (15-17). For normal mouse tissue, the initial formation of γ-H2AX shows a linear dose response at 30-60 min after irradiation. Many studies have used residual damage and γ-H2AX foci at 24 h as a measure of the capacity for double-strand break repair (18, 19). It has also been shown that γ-H2AX foci can be identified 7 days after exposure of mice to ionising radiation (15). However, these data relate to keratinocytes within the epidermis. Studies in tumour patients have looked at intervals of up to 10 days (20, 21). From these, there are confirmatory data on the presence of γ-H2AX foci in irradiated cells at relatively long time intervals after exposure. Given the repair kinetics in irradiated cells and tissues, it has to be assumed that lesions persisting after 7-10 days will remain unrepaired (22). Unrepaired persistent damage is the prerequisite for development of chronic-progressive late normal tissue toxicity. In other words, the cascade of functional and structural tissue changes after irradiation will not start if all immediate radiation-induced damage can be repaired. We provide preliminary evidence that supports a new research hypothesis, namely that monitoring the time course of these foci might allow for prediction of late toxicity and development of protection strategies.
Materials and Methods
This study was performed as part of a larger preclinical research project where mouse kidney and spinal cord were irradiated with the aim of exploring radioprotective strategies (23, 24). In the initial spinal cord experiments, the radiation dose response relationship for myelopathy was explored and in parallel, a pilot study of erythropoietin administration was conducted.
Female adult C3H/N mice (12 weeks old) were purchased from Charles River Laboratories, Sulzfeld, Germany. Animals were housed in conventional rodent facilities at the Department of Experimental Oncology, Klinikum rechts der Isar, Munich, Germany, as described elsewhere (23, 24). These facilities are accredited by the Government of Upper-Bavaria and are operated in accordance with state standards and laws. The Governmental Animal Care and Use Committee approved the experimental protocol. Mice were treated in a prone position with 70 kV X-rays (Philips RT100) to a 1-cm wide single field that included the cervical spinal cord (at a dose rate of approximately 6 Gy per minute and source to skin distance of 10 cm). The animals were anaesthetized during irradiation by inhalation of 1.5-2.0% halothane (plus oxygen 0.5 l/min) using a semi-circuit inhalation anaesthesia system to immobilize them in the desired position. In the main experiment (evaluation of the dose response relationship) graded doses of radiation were administered. The first radiation fraction was kept constant at 17 Gy. Seven days later, the second fraction was administered (lowest dose level 11 Gy, 2-Gy increments). Each dose group included 5-6 mice.
Previous studies had suggested that erythropoietin administration starting immediately before irradiation might reduce the extent of brain necrosis in rats exposed to a single dose of 100 Gy (25). These data provided a rationale for the study of two different erythropoietin doses in the spinal cord model. In parallel, a pilot study was run where 4 additional groups received radiotherapy (17 Gy plus 15 Gy and 17 Gy plus 19 Gy, respectively) and Epoetin alfa (Janssen-Cilag, Neuss, Germany) at two different dose levels (10 IE and 40 IE, respectively). The drug was injected subcutaneously 24 h before, 1 h after and 24 h after each dose of radiation, i.e. for a total of 6 injections per mouse. Follow-up was one year after irradiation. The animals were then sacrificed and the spinal cord of selected mice was examined histologically.
For this purpose, paraffin-embedded histological slides from irradiated spinal cord were prepared. We used all surviving mice that did not develop neurological damage within one year after irradiation with 17+19 Gy, i.e. the dose where the dose-response curve started to bend upward. We assumed that mice treated with high radiation doses would be more likely to harbour unrepaired damage than those treated with lower doses. Slides were prepared from 4 unirradiated control mice, 5 irradiated mice and 5 mice treated with radiation plus two different doses of erythropoietin. Immunohistochemical examination was performed with phospho-histone H2AX (Ser139) antibodies (Cell Signaling Technology, Inc., Danvers, MA, USA). The stained slides were evaluated under a microscope (Olympus BX51, ×40) by counting the stains in 10 high power fields (HPF). The results were compared with the Wilcoxon, Mann-Whitney U-test. All reported p-values are two-sided. A p-value <0.05 was considered statistically significant.
Results
Unexpectedly, none of the animals developed neurological deficits after irradiation with doses between 17+11 Gy and 17 Gy + 17 Gy. Only in the 17+19 Gy groups did two animals develop signs of spinal cord damage. One mouse belonged to the control group and one to the 40 IE erythropoietin-treated group. Thus, the experiment did not allow for computation of a complete radiation dose-response curve, which ranges from 0-100% incidence of myelopathy. In unirradiated spinal cord, γ-H2AX foci were virtually absent. In the group of 5 irradiated mice, a mean of 155 foci were counted (range 45-216, median 205). In the lower dose erythropoietin group, a mean of 98 foci were counted (range 4-187, median 61), p=0.15. In the higher dose erythropoietin group, the mean number was 85 (range 40-123, median 89), p=0.25. Figure 1 shows representative histological slides, demonstrating that the γ-H2AX foci were found predominantly in blood vessel endothelium.
Discussion
As known from previous experiments and confirmed here, the evaluation of radioprotective strategies in late-responding normal tissues such as spinal cord is a very time-consuming process. Even rationally designed experiments might lead to unexpected results necessitating subsequent studies with equally long follow-up. It would thus be helpful to identify surrogate endpoints of radiation myelopathy. Such surrogate endpoints should be analyzable during the clinically asymptomatic latent period, ideally as early as possible. As unrepaired cellular damage is a prerequisite for overt side effects of radiotherapy, we hypothesize that detection of residual damage might predict for myelopathy. We provide immunohistochemical data suggesting that residual damage can be detected in mouse spinal cord. This finding is not unexpected, as other groups previously have identified γ-H2AX foci in different irradiated mouse tissues several days after exposure (15, 18). Our own data also suggest that such foci continue to be detectable over a long time period, which clearly extends into the phase where development of late reactions starts. It is therefore justifiable to study the time course of γ-H2AX detection in greater detail. Such studies might start 30-60 min after irradiation and include groups with intermediate (weeks) and long follow-up (months). It is also necessary to correlate γ-H2AX foci with the primary endpoint, i.e. radiation myelopathy, in animals irradiated to higher doses than the ones used in our experiment. After successful completion of these steps, it can be explored whether a given intervention or radioprotective agent reduces the number of foci and whether this translates into reduced incidence of radiation myelopathy. In this context, it is important to understand that DNA damage plays a crucial role in the development of radiation-induced reactions. However, other types of damage also exist. As these might not contribute to a large extent to radiation-induced myelopathy, the γ-H2AX method appears promising. The erythropoietin data shown in the present report were not statistically different from those of the control group, but the statistical power is low with groups of 5-6 animals. The fact that fewer foci were detectable in erythropoietin-treated mice is compatible with the protective effect observed in a previous study of brain irradiation (25); one should also be aware of the fact that the brain study differs from the present one with regard to longer application of the drug, i.e. 10 days after single-fraction irradiation. Future studies might therefore include prolonged drug treatment.
Representative immunohistochemical results. Irradiated mouse spinal cord (upper left) contains γ-H2AX foci predominantly in the blood vessel endothelium (lower panel). Unirradiated control is shown in the upper right corner.
The most plausible explanation for the unexpectedly high tolerance of the spinal cord to irradiation in our mouse model is that we underestimated the volume effect. We chose the radiation dose levels from previous publications (10-12). However, these authors had irradiated larger spinal cord areas and the irradiated volume is an important determinant of response, besides the radiation dose. Nevertheless, the radiation doses used in our model are adequate for examination of subclinical long-term consequences of radiation.
Current models of radiation-induced CNS changes include a cascade of complex and dynamic interactions between mature parenchymal cells (oligodendrocytes, astrocytes, microglia, neurons), stem and progenitor cells and the vascular system, also resulting in important alterations of the local microenvironment (2, 3, 26). Radiobiological studies of boron neutron-capture therapy (BNCT) support the view that vascular damage is one of the crucial components leading to radiation-induced myelopathy after higher doses (4). By choosing boron compounds which are unable to cross the blood-brain barrier, a largely selective irradiation of the vessel walls can be accomplished with BNCT. Compared to conventional non-selective radiotherapy methods, spinal cord lesions with similar histological appearance were induced. Latency time also was comparable between damage induced by BNCT and conventional radiotherapy. It has also been shown that vasoactive drugs might reduce radiation-induced spinal cord damage (27). These data fit well with our observation that γ-H2AX foci were found predominantly in blood vessel endothelium. In conclusion, we present a hypothesis that might facilitate screening of radioprotective agents and optimisation of dosing with shorter follow-up.
- Received December 1, 2010.
- Revision received January 11, 2011.
- Accepted January 12, 2011.
- Copyright© 2011 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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