The vascular endothelial cell is believed to be a major target cell of radiation-induced injury to the central nervous system. Dysfunction of the blood-brain barrier is associated with radiation-induced white matter lesions. The aim of this study was to determine the role of hypoxia in radiation-induced blood-brain barrier disruption. Adult rats were irradiated with graded single doses of 0–22 Gy to the cervical spinal cord. At various times up to 28 weeks after radiation, blood-spinal cord barrier (BSCB) permeability was assessed using immunohistochemistry with antialbumin antibody and gamma counting of 99mTc-diethylenetriamine pentaacetic acid. Expression of vascular endothelial growth factor (VEGF) was assessed using immunohistochemistry and in situ hybridization. Hypoxia was assessed using two 2-nitroimidazole markers, [125I]iodoazomycin arabinodise and 2-(2-nitro-1H-imidazol-l-yl)-N-(2,2,3,3,3,-pentafluoropropyl) acetamide (EF5), with binding in the rat spinal cord measured using gamma counting and immunohistochemistry, respectively. In the nonirradiated rat spinal cord, there was no evidence of BSCB disruption or VEGF expression. After 16–22 Gy, there was a dose-dependent increase in albumin staining and 99mTc-diethylenetriamine pentaacetic acid activity beginning at 16 weeks, consistent with barrier breakdown. A similar dose-dependent increase in white matter astrocytes that showed immunoreactivity and in situ hybridization signals for VEGF was observed. No increase in VEGF-positive cells was observed in gray matter. By 20 weeks after 20–22 Gy, animals developed white matter necrosis associated with diffuse albumin staining. Irradiated rat spinal cord showed a dose (16–22 Gy)- and time-dependent (16–20 weeks after 22 Gy) increase in [125I]iodoazomycin arabinodise accumulation compared to nonirradiated controls. A similar pattern of dose- and time-dependent EF5 immunoreactivity was also observed in white matter. Areas of EF5 expression and VEGF in situ signals colocalized with areas of albumin immunoreactivity. It is concluded that there is a dose-dependent temporal and spatial association of hypoxia, VEGF up-regulation, and radiation-induced BSCB dysfunction. Hypoxia may provide the signal for VEGF up-regulation and perpetuate endothelial permeability damage in the central nervous system after ionizing radiation.

The CNS4 is one of the major dose-limiting organs in clinical radiotherapy. Manifestations of clinical damage typically develop after a latent period of several months to years and are often associated with devastating neurological sequelae (1). Histological lesions after radiation injury include demyelination, white matter necrosis, vasculopathy, and glial atrophy (2). Oligodendrocytes and vascular endothelial cells have been postulated to be target cells. The specific pathways linked to damage development, however, have remained unknown (3).

The site of the BBB is the endothelium of all intracerebral vessels, except those in certain high permeability regions. The BBB is formed by the vascular endothelial cells and the attachment of astrocytes to the capillary wall (4). The presence of the BBB limits the passage of large molecules and some small molecules from the blood into the CNS parenchyma. After injury to the cerebral vasculature, the damaged vessels allow peptides and proteins to leak from blood stream. Although the importance of the structural integrity and permeability of the microcirculation leading to the CNS clinical radiation syndromes has been documented in many studies (5, 6, 7, 8), the underlying cellular and molecular mechanisms of radiation-induced BBB disruption are not fully understood.

Hypoxia is thought to develop where there are limitations in oxygen supply from the vasculature because of deficient vascularization and local microcirculation disturbances. Hypoxia can occur rapidly during the shutdown of blood flow from ischemic or traumatic injury or much more slowly in cases that arise from various other types of progressive vascular disease. Hypoxia causes a wide range of responses at both the systemic and cellular level and has been proposed to regulate many physiological and pathological processes (5). It is believed to be the crucial environmental stimulus for up-regulation of VEGF (6, 7, 8), which acts to increase the immediate availability of oxygen from capillaries through increased vascular permeability and induce formation of new vessels.

Up-regulation of VEGF was reported recently (9, 10) in the irradiated neonatal and adult CNS. It has been postulated that VEGF plays a role in mediating the vascular permeability changes observed after irradiation. We wanted to assess whether hypoxia plays a role in this pathway of injury. Using the rat radiation myelopathy model (11, 12), we observed a dose- and time-dependent response for hypoxia and its temporal and spatial association with VEGF up-regulation and BSCB disruption in the irradiated spinal cord. Our results are thus consistent with the hypothesis that hypoxia provides the signal for VEGF up-regulation and perpetuates the endothelial damage and permeability changes leading to white matter necrosis after irradiation.

Animals.

Female 9–10-week-old Fischer 344 rats weighing approximately 155 grams were used. They were housed 2 animals/cage, fed a standard rodent diet, and given water ad libitum in the animal colony of the Ontario Cancer Institute, a laboratory animal colony accredited by the Canadian Council of Animal Care. All experimental protocols involving animals were approved by the Institute Animal Care Committee.

Irradiation.

Animals were immobilized during irradiation in a polystyrene foam jig using inhalation anesthesia with halothane. Check films for accuracy of field placement and in vivo thermoluminescence dosimetry were performed before the experiments. A single dose of 8, 16, 17, 19, 20, or 22 Gy was delivered to a 2-cm segment of the spinal cord from C2 to T2 using a parallel opposed pair of 100 kV X-rays. No white matter lesions have been observed after a single dose of 8 Gy (10). A single dose of 17, 19, and 22 Gy represented the ED0, ED50, and ED100, respectively, for forelimb paralysis within 180 days secondary to white matter necrosis in this model (12). Control rats were not irradiated. At least three rats were tested at each experimental time point, unless otherwise indicated.

Histopathology.

Animals were anesthetized with i.m. ketamine (Vetrepharm) at 0.1 ml/100 grams of body weight and Atravet (Ayerst) at 0.05 ml/100 grams of body weight. After a 1-min transcardiac perfusion of 0.9% saline to clear blood from the circulatory system, animals were perfused with a 10-min transcardiac infusion of 10% buffered neutral formalin. The spinal cord was dissected out, and sections were obtained by cutting the spinal cord transversely at midplane between C2 and T2. Specimens were processed using a standard paraffin-embedding method within 48–72 h for routine H&E and immunohistochemical studies. For in situ hybridization, sections were cut at 5-μm thickness and mounted on Super Frost/Plus microscopic slides (Fisher Scientific).

For assessment of hypoxia using EF5, animals were injected i.v. with 1.0 ml of 10 mm EF5 (a gift from Dr. C. Koch, University of Pennsylvania, Philadelphia, PA) in 0.9% saline/100 grams of body weight. Anesthetized animals were sacrificed at 3 h after EF5 injection, and the spinal cord was dissected out, frozen immediately in liquid nitrogen, and embedded in OCT compound (Sacura Finetek). A series of cryostat sections of 10-μm thickness was obtained by cutting the spinal cord transversely at midplane between C2 and T2.

Immunohistochemistry.

VEGF protein expression after irradiation was examined immunohistochemically using a rabbit polyclonal antirat VEGF antibody (VEGF-147; Santa Cruz Biotechnology). The integrity of the BSCB was studied using a sheep polyclonal antirat albumin antibody (Cedarlane) with immunostaining of albumin in the interstitium as a surrogate for disruption of the BSCB (10, 13). Immunohistochemistry for VEGF and albumin was performed using the streptavidin-HRP-biotin technique as described previously (10, 14). Briefly, sections were deparaffinized and blocked in 3% H2O2 and 5% normal goat serum, followed by incubation with the respective primary antibodies (1:200 dilution for VEGF and 1:6400 dilution for albumin). This was followed by incubation with biotinylated linking antibody and HRP (Dako), with brief rinses in PBS between incubations. The reaction was visualized using 3-amino-9-ethycarbazole (Vector Laboratories) and counterstained with Mayer’s hematoxylin. Reagent controls (omitting the primary antibody or substituting nonimmune serum for the primary antibody in the staining protocol) on tissue sections revealed no staining, thus confirming the specificity of the primary antibodies used.

For EF5 immunohistochemistry, cryostat sections were fixed with 10% formalin for 10 min. After washing in PBS, sections were incubated for 1 h with the monoclonal antibody ELK3-51 conjugated with biotin (a gift from Dr. C. Koch, University of Pennsylvania, Philadelphia, PA). The slides were then washed in PBS and incubated in streptavidin conjugated to alkaline phosphatase (Dako) for 30 min. After washes in PBS, sections were incubated with New Fuchsin (New Fuchsin Kit; Dako) for 30 min and counterstained with hematoxylin.

To determine whether there was spatial colocalization of areas of hypoxia, BSCB breakdown, and VEGF mRNA expression, the cryostat sections were stained for EF5 or albumin (Dako) immunohistochemically, and in situ hybridization for VEGF was processed on adjacent sections.

In Situ Hybridization.

In situ hybridization was performed as described previously (10). The pair of primers used was previously shown to detect VEGF121, VEGF165, and VEGF189 in the normal rat CNS (15, 16). The plasmid carrying a 403-bp fragment of the rat VEGF cDNA coding sequence was linearized with XhoI (Boehringer Mannheim), and the orientation was confirmed by restriction site analysis using PstI (Boehringer Mannheim). The identity of the DNA fragment was confirmed by DNA sequence. The DIG-labeled sense and antisense RNA probes were obtained using a DIG RNA labeling kit (Boehringer Mannheim) according to the manufacturer’s instructions. Briefly, the slides were fixed in 4% paraformaldehyde in PBS and treated with 0.2 n HCl and proteinase K. The sections were acetylated in freshly prepared 0.25% acetic anhydride in 0.1 m triethanolamine-HCl (pH 8.0). The slides were prehybridized in 50% formamide, 5× SSC, 5× Denhardt’s solution, 250 μg/ml yeast tRNA, and 500 μg/ml salmon sperm DNA (buffer 1). Hybridization with DIG-labeled sense or antisense probes (100 ng/100 μl) in buffer 1 was performed at 70°C, followed by posthybridization wash in 0.2× SSC at 70°C. The slides were then equilibrated in 0.2× SSC, 0.1 m Tris (pH 7.5), and 0.15 m NaCl (buffer 2) before blocking in 10% fetal bovine serum/buffer 2. This was followed by incubation of anti-DIG antibody conjugated with alkaline phosphatase (Boehringer Mannheim) and diluted 1:500 in 10% fetal bovine serum/buffer 2. Visualization of the VEGF mRNA message was done by incubating sections in 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium. The slides were counterstained with methyl green (Sigma). Sections of rat lung were used as a positive control.

Assessment of Hypoxia and BSCB Permeability.

The hypoxia tracer [125I]IAZA, the BBB/BSCB permeability tracer 99mTc-DTPA, and the blood flow tracers 99mTc-ECD and 99mTc-HMPAO were used to assess hypoxia, BSCB breakdown, and perfusion, respectively, in the rat spinal cord after irradiation. [125I]IAZA was prepared by exchange labeling of IAZA precursor with [125I]sodium iodide (NEZ-033; New England Nuclear Life Science Products Inc., Boston, MA) as described previously (17) and purified on a solid-phase extraction cartridge (18). The IAZA precursor 1-(5&′-iodo-5&′-deoxy-β-d-arabinofuranosyl)-2-nitroimidazole was donated by Dr. L. Wiebe, University of Alberta, Edmonton, Canada. 99mTc-DTPA, 99mTc-ECD, and 99mTc-HMPAO were prepared by the addition of 200 MBq of Na99mTcO4 (Technelite generator; DuPont Pharma, Billerica, MA) and saline to kits of DTPA-Plus (DraxImage, Montreal, Quebec, Canada), Neurolite (DuPont Pharma), and Ceretec (Nycomed Amersham, Amersham, United Kingdom), respectively.

At intervals between 14 and 20 weeks after 22 Gy and at 20 weeks after 16, 19, or 22 Gy, rats were injected i.p. with 0.4 MBq of [125I]IAZA and i.v. with 4 MBq of one of the 99mTc-labeled tracers, respectively. Two h later, the rats were sacrificed, and 1 ml of blood sample was obtained from the heart, and the segment of the spinal cord (C2-T2) was dissected. Blood and the spinal cord samples were weighed and counted for 99mTc immediately along with a standard in a gamma counter. Assay for 125I was performed 1 week later to allow decay of 99mTc along with a standard. Results were expressed as the percentage of the injected dose/gram tissue and spinal cord:blood ratios.

All data represent the mean ± SE, unless otherwise indicated. Using the Mann-Whitney test, a nonparametric test, each group of animals at each time or dose point was compared with the group of nonirradiated controls to determine whether the percentage of the injected dose/gram of tissue and spinal cord:blood ratios were significantly different.

Histopathological Changes after Irradiation

Routine histological examination of the rat spinal cord was performed on H&E-stained sections between 16 and 28 weeks after graded single doses of 0–22 Gy of irradiation. After a single dose of 8 Gy, the spinal cord remained histologically normal with no evidence of any histopathological changes. Similarly, there were no apparent histopathological abnormalities or white matter or blood vessel changes after a single dose of 16, 17, or 18 Gy. After a single dose of 19, 20, or 22 Gy, histopathological changes were not observed in the rat spinal cord until 19 and 20 weeks, when focal to extensive necrosis and varying degrees of demyelination appeared in white matter. The lesions consisted of randomly distributed, discrete areas of cell loss, necrotic debris, swollen axons, and vacuoles in white matter. Hemorrhage was occasionally observed. There was a notable absence of inflammatory infiltrate. No gross vascular lesions were apparent. No lesions were observed in gray matter.

Hypoxia in the Irradiated Rat Spinal Cord

EF5 Immunohistochemistry.

Hypoxia was assessed immunohistochemically using EF5 at 20, 24, and 28 weeks after a single dose of 17 Gy and at 16, 17, 18, 19, 20, 21, and 22 weeks after a single dose of 22 Gy. No EF5 immunostaining was observed in nonirradiated age-matched controls (Fig. 1,A) or in the rat spinal cord irradiated with a single dose of 22 Gy without EF5 injection (Fig. 1,B). Reagent controls with the primary antibody omitted revealed no staining and thus confirmed the specificity of the primary antibody used (data not shown). Occasional focal EF5 immunostaining in white matter was seen at 20 (Fig. 1,C), 24 (Fig. 1,D), or 28 weeks (Fig. 1,E) after a single dose of 17 Gy, and there was no relation between the time interval after 17 Gy and the size of the EF5 staining area. No EF5 staining was observed at 16 (Fig. 1,F), 17, or 18 weeks in the rat spinal cord after 22 Gy. Varying extents of EF5 staining were observed at 19 weeks (Fig. 1,G), which increased dramatically by 20 weeks after 22 Gy. Where white matter necrosis was observed (20–22 weeks after 22 Gy), EF5 staining was seen in cells surrounding areas of necrosis (Fig. 1 H). There was no evidence of staining in control or irradiated gray matter.

[125I]IAZA Uptake.

Evidence for hypoxia after irradiation was assessed and quantified using a second method, namely, using [125I]IAZA as a hypoxia tracer. In nonirradiated age-matched controls, the absolute accumulation of [125I]IAZA in rat spinal cord was 0.06 ± 0.01% injected dose/gram of tissue. The accumulation in the rat spinal cord between 14 and 15 weeks after 22 Gy was similar to that seen in controls. After 22 Gy, the spinal cord showed an increase in [125I]IAZA accumulation beginning at 16 weeks (0.09 ± 0.01%) compared to nonirradiated age-matched controls (P = 0.02; Fig. 2, top left panel). Blood levels of [125I]IAZA at 2 h after injection were 0.17 ± 0.05% dose/gram and did not vary among groups. Thus, spinal cord:blood ratios showed the same pattern as absolute uptake in spinal cord (data not shown). At 20 weeks after a single dose of 16, 19, or 22 Gy, a radiation dose-response was evident in both the accumulation of [125I]IAZA in the spinal cord (Fig. 2, bottom left panel) and the spinal cord:blood ratio (data not shown).

BSCB Breakdown after Irradiation

99mTc-DTPA Uptake.

The BSCB disruption after irradiation was assessed by measuring 99mTc-DTPA penetration. The absolute accumulation of 99mTc-DTPA increased with time after 22 Gy in the spinal cord (Fig. 2, top middle panel), starting at a similar time as the [125I]IAZA increases. Blood levels of 99mTc-DTPA at 2 h after injection were 0.020 ± 0.005% dose/gram and did not vary among groups; thus, spinal cord:blood ratios showed the same pattern as absolute uptake in spinal cord (data not shown). A radiation dose-response effect was evident in the rat spinal cord at 20 weeks after a single dose of 16, 19, or 22 Gy (Fig. 2, bottom middle panel).

Perfusion in the spinal cord was measured using 99mTc-HMPAO and 99mTc-ECD, both of which are trapped in viable cells. As a function of time after a single dose of 22 Gy, 99mTc-HMPAO accumulation increased at 17 weeks and reached a plateau from 18–20 weeks (P < 0.04) compared to control (Fig. 2, top right panel). No apparent radiation dose-response effect was evident for 99mTc-ECD. Accumulation in the rat spinal cord at 20 weeks after 16 or 19 Gy was much greater than that seen in controls (P = 0.02), whereas accumulation after 22 Gy, although still higher than that in controls, was less than that at 16 or 19 Gy (Fig. 2, bottom right panel).

Albumin Immunohistochemistry.

The integrity of the BSCB was also assessed using immunostaining for albumin in the CNS parenchyma as a surrogate for disruption of the BSCB. In the nonirradiated rat spinal cord (Fig. 1,I) and after a single dose of 8 Gy irradiation (Fig. 1,J), there was no evidence of albumin immunoreactivity. After a single dose of 17 or 18 Gy (Fig. 1,K), there was occasional focal staining for albumin seen at 20 weeks in white matter. At 20 weeks, diffuse albumin staining was seen after a single dose of 19 Gy (Fig. 1,L), and extensive staining was observed after 20 Gy (Fig. 1 M).

After a single dose of 22 Gy, focal albumin immunostaining in white matter consistent with breakdown of BSCB was first observed at 16 weeks. By 18 weeks, diffuse staining was often observed filling up the entire white matter compartment, and at 20 weeks, when white matter necrosis developed, there was extensive staining seen in both white (Fig. 1 N) and gray matter.

VEGF Up-Regulation after Irradiation

VEGF Immunohistochemistry.

In the nonirradiated rat spinal cord, there was little evidence of VEGF immunoreactivity, regardless of the age of the animals (Fig. 3 A). After a single dose of 8 or 17 Gy, there was also no apparent increase in the percentage of VEGF-immunoreactive cells in white and gray matter up to 28 weeks compared with nonirradiated controls.

After a single dose of 22 Gy, an increase in white matter glial cells that showed positive immunostaining for VEGF was first observed at 16 weeks. By 20 weeks after 22 Gy, there was a marked increase in VEGF-positive cells in white matter (Fig. 3 B). VEGF-positive cells were often observed around areas of necrosis. There was no evidence of an increase in the percentage of VEGF-positive cells in gray matter after a single dose of 22 Gy. VEGF immunostaining appeared confined to glial cells and was not observed in neurons or vascular endothelial cells.

VEGF in Situ Hybridization.

VEGF mRNA expression was not observed in nonirradiated age-matched rat spinal cord (Fig. 3,C). After single doses of 8–18 Gy (Fig. 3,D), there was no evidence of cells positive for VEGF in situ hybridization in the rat spinal cord up to 20 weeks. After single doses of 19–22 Gy, focal VEGF mRNA signals were first observed at 16 weeks, with a marked increase in the number of VEGF mRNA-positive cells observed by 20 weeks (Fig. 3,E). Expression of VEGF mRNA at 20 weeks after graded single doses of 0–22 Gy is shown in Fig. 4. The specificity of the VEGF mRNA signals was confirmed by the absence of staining in sense probe controls (Fig. 3 F).

Cell Type-specific Up-Regulation of VEGF.

Signals for mRNA were seen only in glial cells and not in neurons and vascular endothelial cells. At 20 weeks after 22 Gy, VEGF in situ hybridization combined with GFAP immunofluorescence staining revealed that the majority of VEGF-expressing cells were astrocytes (74.8 ± 2.3%; Fig. 3, G and H). The occasional VEGF-positive cells (3.8 ± 0.6%) showed immunoreactivity for the microglial marker lectin RCA-1 (Fig. 3, I and J).

Colocalization of EF5, Albumin, and VEGF mRNA.

Colocalization of EF5, albumin immunostaining, and VEGF mRNA expression was assessed using adjacent 10-μm-thick frozen sections in the rat spinal cord at 21 weeks after 22 Gy. EF5 immunostaining was localized mainly in cells surrounding areas of necrosis in white matter (Fig. 3,K), and a similar staining pattern was also seen using albumin (Fig. 3,L). Signals for VEGF mRNA were prominently detected in reactive glial cells surrounding white matter necrosis (Fig. 3 M).

Microvessel endothelial cells in the CNS are major structural and functional components of the BBB that maintain the homeostasis of the CNS. In the present study, BSCB integrity was assessed using two different methods, immunohistochemistry for endogenous albumin (13) and gamma counting of 99mTc-DTPA given i.v. as a vascular tracer. Albumin in the CNS is normally restricted to the vasculature. DTPA is a water-soluble tracer. It is normally excluded from the brain and spinal cord but accumulates nonspecifically where there are defects in the BBB and BSCB (19). Results from the albumin and DTPA experiments both indicated that BSCB integrity was disturbed beginning at 16 weeks after 22 Gy. This is consistent with our previous study in which HRP was used as a BSCB marker (20). Accumulation of 99mTc-DTPA showed a dramatic increase as a function of time after irradiation and a radiation dose-response relationship in the rat spinal cord. This was particularly evident because of the extremely low accumulation in nonirradiated spinal cord. These results again parallel those obtained with albumin immunohistochemistry.

99mTc-HMPAO and 99mTc-ECD are used clinically as tracers of cerebral perfusion (21, 22). Both are neutral, lipophilic chelates that cross the intact BBB by passive diffusion, rapidly undergo a chemical change, and are unable to diffuse out of the brain and thus trapped in proportion to regional perfusion. The mechanism of trapping of the two tracers differs. 99mTc-HMPAO undergoes nucleophilic attack from intracellular glutathione (21), whereas 99mTc-ECD is a substrate for nonspecific esterases (22). In the brain, accumulation of the tracers in gray matter is 3-fold higher than that in white matter, in proportion to the difference in perfusion. In the present study, 99mTc-HMPAO accumulation in the rat spinal cord after 22 Gy was greater than that in control from week 17 but essentially reached a plateau from weeks 18–20. An analogous pattern was seen in the radiation dose-response experiment with 99mTc-ECD, where the 16- and 19-Gy doses resulted in 99mTc-ECD accumulation that was four times greater than that in the control ,whereas the 22 Gy levels were only 3-fold greater than that of the control. An increase in perfusion and metabolism in the human spinal cord after radiation has been reported (23). Increased permeability of the BSCB may allow penetration of hydrophilic metabolites that constitute the major proportion of the radioactivity in the blood. In the present study, this is unlikely to be an artifact of increased permeability because the extent at earlier times and after lower radiation doses was far greater than that seen with 99mTc-DTPA. The observation of a similar effect with two tracers having different mechanisms of retention also suggests that the effect is real. The lack of a further increase in accumulation of radioactivity at later times and higher doses is easier to explain. This is likely due to the combined effects of a decreased number of viable cells to trap the tracers and an increased permeability of the BSCB, allowing the hydrophilic metabolites to exit the spinal cord.

[125I]IAZA and EF5 are both 2-nitroimidazoles. Their characteristic reduction and binding to cellular macromolecules in the absence of oxygen allows their use for the detection and characterization of hypoxia (21, 24). When IAZA is labeled with 123I, its distribution in the body can be imaged noninvasively using single-photon emission computed tomography. IAZA has been used extensively in the detection of hypoxia in tumors (25, 26). A combination of 99mTc-HMPAO and [125I]IAZA has been used to study cerebral ischemia in rat brain (27). In the present study, [125I]IAZA accumulation in the spinal cord increased with time after irradiation, becoming significantly higher than that in the control by 16 weeks after 22 Gy. IAZA levels in the irradiated spinal cord were 2.5-fold greater than those in controls, and [125I]IAZA accumulation also showed a radiation dose-response at 20 weeks. The increase in IAZA accumulation seen before white matter necrosis became evident histologically. This is consistent with the involvement of hypoxia in this pathway of radiation injury.

EF5 is a pentafluorinated derivative of the 2-nitroimidazole etanidazole (24). Its mechanism of binding to hypoxic tissues is the same as that of IAZA, although the method of detection differs. No EF5 expression was observed at 16 or 18 weeks after 22 Gy, suggesting that the radioactive tracer technique is a more sensitive approach than immunohistochemistry using EF5. The molecular weight of IAZA (355.1) is quite similar to that of EF5 (Mr 302; Ref. 26), suggesting that the difference is merely the detection limit for radioactivity being lower than that for immunohistochemistry. At 20 weeks, EF5 expression in rats that received 16 Gy was similar to that in controls, whereas those that received 19 and 22 Gy showed progressively greater accumulation. We were thus able to demonstrate a radiation dose-response effect using both the radioactive and immunohistochemistry methods.

In the irradiated rat spinal cord, tissue hypoxia may result in increased blood flow and total blood volume, as we observed in the radiotracer experiments. Increased blood flow in the irradiated rat brain has been reported previously (28, 29). These findings are consistent with quantitative histological studies, which demonstrated the dilation of microvessels and an increase in vascular volume in the rat spinal cord at late times after irradiation (20, 30). The increase in ECD or HMPAO accumulation may thus be related in part to the increased area of blood vessel wall available to the tracers and hence to an increase in spinal cord blood flow or perfusion.

VEGF is a potent angiogenic and endothelial cell-specific mitogen. VEGF and its receptors are up-regulated during embryonic and early postnatal brain development but down-regulated in the adult CNS when angiogenesis ceases (31, 32). Hypoxia was found to be a potent inducer of VEGF expression in several organs including the CNS (33). In tumors, VEGF is localized in the most ischemic and necrotic areas of the tissues, consistent with the notion that local hypoxia is a potent inducer of VEGF production (6). The observation of VEGF up-regulation thus provides further evidence for the presence of hypoxia associated with radiation-induced breakdown of BBB or BSCB.

VEGF is also known as vascular permeability factor. It was originally found to induce hyperpermeability in vessels to circulating macromolecules (34). Its expression is increased in brain tumors associated with leaky vasculature (35). In rat retinas, hyperpermeability of microvessels was associated with VEGF immunoreactivity (36), and VEGF increased the permeability of the cultured bovine brain endothelial cells that express BBB characteristics (37). The mechanisms by which VEGF affects vessel permeability are not known, but VEGF has been shown to induce fenestrations in capillary endothelial cells (38) and to affect occludin expression and tight junction assembly (39). We postulate that VEGF is involved in increasing the permeability of the vasculature in the irradiated rat spinal cord and thus perpetuates the endothelial cell damage.

Consistent with previous observations (10), dual immunostaining of VEGF mRNA-expressing cells with GFAP or lectin RCA-1 indicated that the majority of these cells were astrocytes. In the adult spinal cord, astrocyte but not vascular endothelial cell proliferation has been described in association with VEGF up-regulation and BSCB disruption after a single dose of 22 Gy (10). In the neonatal rat spinal cord irradiated to a very high dose of 55 Gy, VEGF up-regulation was also observed in astrocytes (9). In a rodent model of cerebral infract, macrophages, microglia (40), and neurons (41, 42) have been described to be the major VEGF-expressing cells. Thus, up-regulation of VEGF associated with tissue hypoxia/ischemia appears to be cell type specific and damage specific.

In summary, the present study showed that there was a dose-dependent temporal and spatial association of hypoxia and VEGF up-regulation with radiation-induced BSCB dysfunction in the rat spinal cord. We propose that ionizing radiation induces endothelial cell damage and subsequent BSCB breakdown. This leads to vasogenic edema and hypoxia. Hypoxia induces VEGF expression in reactive astrocytes, which in turn leads to further increase in vascular permeability and BSCB disruption. VEGF has been shown to induce adhesion molecules such as intercellular adhesion molecule 1 on endothelial cells and modulate inflammation in rat brain (43). The present data are thus consistent with a dynamic process of BCSB disruption, vasogenic edema, hypoxia, cytokine responses, and secondary injury in the CNS resulting in white matter necrosis (Refs. 44 and 45; Fig. 5). Targeting hypoxia and/or VEGF may provide a basis on which BBB disruption after ionizing radiation can be modulated and thus offers a therapeutic strategy to protect the CNS from radiation damage.

Fig. 1.

Time course of the hypoxia marker, EF5, uptake in the rat spinal cord after a single dose of 17 or 22 Gy. No EF5 staining was observed in nonirradiated age-matched controls (A) and in irradiated (22 Gy) rat spinal cord without EF5 injection (B). Occasional focal EF5 staining was observed at 20 (C), 24 (D), or 28 weeks (E) after 17 Gy. No EF5 staining was observed at 16 weeks (F) after 22 Gy. Focal to extensive EF5 staining was observed at 19 weeks (G), which increased dramatically at 20 weeks after 22 Gy (H) in an animal in which EF5 staining is seen surrounding an area of necrosis (∗) in white matter. Dose-response for BSCB breakdown assessed by immunohistochemistry using albumin at 20 weeks after a single dose of 8, 17, 18, 19, 20, or 22 Gy. In nonirradiated age-matched controls (I) and the irradiated rat spinal cord after 8 Gy (J), no albumin staining was evident. Focal albumin staining was observed after 17 or 18 Gy (K). Diffuse staining was seen after 19 Gy (L), and extensive albumin staining was seen after 20 Gy (M) or 22 Gy (N), when white matter necrosis was also observed. Original magnifications, ×100.

Fig. 1.

Time course of the hypoxia marker, EF5, uptake in the rat spinal cord after a single dose of 17 or 22 Gy. No EF5 staining was observed in nonirradiated age-matched controls (A) and in irradiated (22 Gy) rat spinal cord without EF5 injection (B). Occasional focal EF5 staining was observed at 20 (C), 24 (D), or 28 weeks (E) after 17 Gy. No EF5 staining was observed at 16 weeks (F) after 22 Gy. Focal to extensive EF5 staining was observed at 19 weeks (G), which increased dramatically at 20 weeks after 22 Gy (H) in an animal in which EF5 staining is seen surrounding an area of necrosis (∗) in white matter. Dose-response for BSCB breakdown assessed by immunohistochemistry using albumin at 20 weeks after a single dose of 8, 17, 18, 19, 20, or 22 Gy. In nonirradiated age-matched controls (I) and the irradiated rat spinal cord after 8 Gy (J), no albumin staining was evident. Focal albumin staining was observed after 17 or 18 Gy (K). Diffuse staining was seen after 19 Gy (L), and extensive albumin staining was seen after 20 Gy (M) or 22 Gy (N), when white matter necrosis was also observed. Original magnifications, ×100.

Close modal
Fig. 2.

Time course of accumulation for the hypoxia tracer [125I]IAZA (top left panel), the BBB permeability tracer 99mTc-DTPA (top middle panel), and the perfusion tracer 99mTc-HMPAO (top right panel) in the rat spinal cord at 14–20 weeks after a single dose of 22 Gy (▴) or in nonirradiated age-matched controls (○). Radiation dose-response relationship of accumulation in the rat spinal cord at 20 weeks after a single dose of 0 (○), 16, 19, or 22 Gy (▴) for hypoxia tracer [125I]IAZA (bottom left panel), BBB permeability tracer 99mTc-DTPA (bottom middle panel), and perfusion tracer 99mTc-ECD (bottom right panel) is shown. Results are expressed as the percentage of injected dose/gram of spinal cord. Each data point represents measurement from an individual animal.

Fig. 2.

Time course of accumulation for the hypoxia tracer [125I]IAZA (top left panel), the BBB permeability tracer 99mTc-DTPA (top middle panel), and the perfusion tracer 99mTc-HMPAO (top right panel) in the rat spinal cord at 14–20 weeks after a single dose of 22 Gy (▴) or in nonirradiated age-matched controls (○). Radiation dose-response relationship of accumulation in the rat spinal cord at 20 weeks after a single dose of 0 (○), 16, 19, or 22 Gy (▴) for hypoxia tracer [125I]IAZA (bottom left panel), BBB permeability tracer 99mTc-DTPA (bottom middle panel), and perfusion tracer 99mTc-ECD (bottom right panel) is shown. Results are expressed as the percentage of injected dose/gram of spinal cord. Each data point represents measurement from an individual animal.

Close modal
Fig. 3.

VEGF expression by immunohistochemistry and in situ hybridization in the rat spinal cord. In the age-matched nonirradiated rat spinal cord (A; original magnification, ×100) and up to 28 weeks after 17 Gy, there was little evidence of VEGF immunoreactivity. A dramatic increase in white matter glial cells that showed positive (red) staining for VEGF was observed at 20 weeks after 22 Gy (B; original magnification, ×100). There was little evidence of VEGF mRNA-positive cells in the nonirradiated rat spinal cord (C) or after single doses of 8–18 Gy (D). Extensive VEGF mRNA signals were found at 20 weeks after 20 Gy (E). Only methyl green-stained cells are seen using a sense probe (F; asterisk denotes an area of necrosis in white matter necrosis; original magnifications in C–F, ×400). VEGF mRNA-expressing cells (G, arrow) by in situ hybridization in white matter necrosis of the rat spinal cord at 20 weeks after 22 Gy are also positive for GFAP immunofluorescence staining (H, arrow). An occasional VEGF in situ hybridization-positive cell (I, arrow) is positive for RCA-1 immunofluorescence staining (J, arrow; original magnifications in G–J, ×1000). Colocalization of EF5, albumin immunostaining, and VEGF mRNA expression was assessed in the rat spinal cord at 21 weeks after 22 Gy using adjacent 10-μm-thick sections. Immunostaining for EF5 (K) is observed in reactive glial cells in an area of white matter that also shows positive staining for albumin (L). These glial cells also show signals for VEGF mRNA (purple staining, M); original magnifications in K–M, ×100.

Fig. 3.

VEGF expression by immunohistochemistry and in situ hybridization in the rat spinal cord. In the age-matched nonirradiated rat spinal cord (A; original magnification, ×100) and up to 28 weeks after 17 Gy, there was little evidence of VEGF immunoreactivity. A dramatic increase in white matter glial cells that showed positive (red) staining for VEGF was observed at 20 weeks after 22 Gy (B; original magnification, ×100). There was little evidence of VEGF mRNA-positive cells in the nonirradiated rat spinal cord (C) or after single doses of 8–18 Gy (D). Extensive VEGF mRNA signals were found at 20 weeks after 20 Gy (E). Only methyl green-stained cells are seen using a sense probe (F; asterisk denotes an area of necrosis in white matter necrosis; original magnifications in C–F, ×400). VEGF mRNA-expressing cells (G, arrow) by in situ hybridization in white matter necrosis of the rat spinal cord at 20 weeks after 22 Gy are also positive for GFAP immunofluorescence staining (H, arrow). An occasional VEGF in situ hybridization-positive cell (I, arrow) is positive for RCA-1 immunofluorescence staining (J, arrow; original magnifications in G–J, ×1000). Colocalization of EF5, albumin immunostaining, and VEGF mRNA expression was assessed in the rat spinal cord at 21 weeks after 22 Gy using adjacent 10-μm-thick sections. Immunostaining for EF5 (K) is observed in reactive glial cells in an area of white matter that also shows positive staining for albumin (L). These glial cells also show signals for VEGF mRNA (purple staining, M); original magnifications in K–M, ×100.

Close modal
Fig. 4.

Expression of VEGF mRNA by in situ hybridization in the rat spinal cord at 20 weeks after single doses of 0–22 Gy. Each data point represents the mean number of VEGF-positive cells observed per transverse section of the spinal cord in three animals (3 sections/animal); bars, SE.

Fig. 4.

Expression of VEGF mRNA by in situ hybridization in the rat spinal cord at 20 weeks after single doses of 0–22 Gy. Each data point represents the mean number of VEGF-positive cells observed per transverse section of the spinal cord in three animals (3 sections/animal); bars, SE.

Close modal
Fig. 5.

Model of CNS radiation injury. Endothelial cell death or damage leads to BBB/BSCB disruption, vasogenic edema, vascular compromise, and, hence, tissue hypoxia. In response to hypoxia, astrocytes produce VEGF that further compromises BSCB integrity. This may trigger an avalanche effect, resulting in white matter necrosis.

Fig. 5.

Model of CNS radiation injury. Endothelial cell death or damage leads to BBB/BSCB disruption, vasogenic edema, vascular compromise, and, hence, tissue hypoxia. In response to hypoxia, astrocytes produce VEGF that further compromises BSCB integrity. This may trigger an avalanche effect, resulting in white matter necrosis.

Close modal

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

Supported by the National Cancer Institute of Canada (C. S. W.) and the Agnico-Eagle Research Fund (C. S. W.).

4

The abbreviations used are: CNS, central nervous system; BBB, blood-brain barrier; VEGF, vascular endothelial growth factor; BSCB, blood-spinal cord barrier; EF5, 2-(2-nitro-1H-imidazol-l-yl)-N-(2,2,3,3,3,-pentafluoropropyl) acetamide; HRP, horseradish peroxidase; DIG, digoxigenin; IAZA, iodoazomycin arabinodise; DTPA, diethylenetriamine pentaacetic acid; ECD, ethyl cysteinate diethylester; HMPAO, hexamethylpropylene amine oxime; GFAP, glial fibrillary acidic protein; RCA-1, Ricinus communis agglutinin-1.

We thank J. Ho for technical assistance and Drs. R. Reilly and C. Koch for helpful suggestions. We are grateful to Dr. L. Wiebe for providing the IAZA precursor and to Dr. C. Koch for EF5 and antibody ELK3-51.

1
Schultheiss T. E., Kun L. E., Ang K. K., Stephens L. C. Radiation response of the central nervous system.
Int. J. Radiat. Oncol. Biol. Phys.
,
31
:
1093
-1112,  
1995
.
2
Schultheiss T. E., Stephens L. C., Maor M. H. Analysis of the histopathology of radiation myelopathy.
Int. J. Radiat. Oncol. Biol. Phys.
,
27
:
27
-32,  
1988
.
3
Hopewell J. W., van der Kogel A. J. Pathophysiological mechanisms leading to the development of late radiation-induced damage to the central nervous system.
Front. Radiat. Ther. Oncol.
,
33
:
265
-275,  
1999
.
4
Rubin L. L., Staddon J. M. The cell biology of the blood-brain barrier.
Annu. Rev. Neurosci.
,
22
:
11
-28,  
1999
.
5
Bunn H. F., Poyton R. O. Oxygen sensing and molecular adaptation to hypoxia.
Physiol. Rev.
,
76
:
839
-885,  
1996
.
6
Shweiki D., Itin A., Soffer D., Keshet E. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis.
Nature (Lond.)
,
359
:
843
-845,  
1992
.
7
Ikeda E., Achen M. G., Breier G., Risau W. Hypoxia-induced transcriptional activation and increased mRNA stability of vascular endothelial growth factor in C6 glioma cells.
J. Biol. Chem.
,
270
:
19761
-19766,  
1995
.
8
Levy A. P., Levy N. S., Wegner S., Goldberg M. A. Transcriptional regulation of the rat vascular endothelial growth factor gene by hypoxia.
J. Biol. Chem.
,
270
:
13333
-13340,  
1995
.
9
Bartholdi D., Rubin B. P., Schwab M. E. VEGF mRNA induction correlates with changes in the vascular architecture upon spinal cord damage in the rat.
Eur. J. Neurosci.
,
9
:
2549
-2560,  
1997
.
10
Tsao M. N., Li Y. Q., Lu G., Xu Y., Wong C. S. Upregulation of vascular endothelial growth factor is associated with radiation-induced blood-spinal cord barrier breakdown.
J. Neuropathol. Exp. Neurol.
,
58
:
1051
-1060,  
1999
.
11
Van der Kogel A. J. Central nervous system radiation injury in small animal models Gutin P. H. Leibel S. A. Sheline G. E. eds. .
Radiation Injury to the Nervous System
,
:
91
-111, Raven Press New York  
1991
.
12
Wong C. S., Minkin S., Hill R. P. Linear-quadratic model underestimates sparing effect of small doses per fraction in rat spinal cord.
Radiother. Oncol.
,
23
:
176
-184,  
1992
.
13
Albayrak S., Zhao Q., Siesjo B. K., Smith M. L. Effect of transient focal ischemia on blood-brain barrier permeability in the rat: correlation to cell injury.
Acta Neuropathol. (Berl.)
,
94
:
158
-163,  
1997
.
14
Preissner C. M., Klee G. G., Krco C. J. Nonisotopic “sandwich” immunoassay of thyroglobulin in serum by the biotin-streptavidin technique: evaluation and comparison with an immunoradiometric assay.
Clin. Chem.
,
34
:
1794
-1798,  
1988
.
15
Lindgren M., Johansson M., Sandstrom J., Jonsson Y., Bergenheim A. T., Henriksson R. VEGF and tPA co-expressed in malignant glioma.
Acta Oncol.
,
36
:
615
-618,  
1997
.
16
Houck K. A., Ferrara N., Winer J., Cachianes G., Li B., Leung D. W. The vascular endothelial growth factor family: identification of a fourth molecular species and characterization of alternative splicing of RNA.
Mol. Endocrinol.
,
5
:
1806
-1814,  
1991
.
17
Mannan R. H., Somayaji V. V., Lee J., Mercer J. R., Chapman J. D., Wiebe L. I. Radioiodinated 1-(5-iodo-5-deoxy-β-d-arabinofuranosyl)-2-nitroimidazole (iodoazomycin arabinoside: IAZA): a novel marker of tissue hypoxia.
J. Nucl. Med.
,
32
:
1764
-1770,  
1991
.
18
Stringer R. E., Wiebe L. I., McEwan A. J. B., Maltby P. J. A simple method for purification and preparation of I-123 azomycin arabinoside (IAZA) for patient use.
Eur. J. Nucl. Med.
,
24
:
988
1997
.
19
Prato F. S., Wisenberg G., Marshall T. P., Uksik P., Zabel P. Comparison of the biodistribution of gadolinium-153 DTPA and technetium-99m DTPA in rats.
J. Nucl. Med.
,
29
:
1683
-1687,  
1988
.
20
Stewart P. A., Vinters H. V., Wong C. S. Blood-spinal cord barrier function and morphometry after single doses of x-rays in rat spinal cord.
Int. J. Radiat. Oncol. Biol. Phys.
,
32
:
703
-711,  
1995
.
21
Ballinger J. R., Reid R. H., Gulenchyn K. Y. Technetium-99m HM-PAO stereoisomers: differences in interaction with glutathione.
J. Nucl. Med.
,
29
:
1998
-2000,  
1988
.
22
Walovitch R. C., Cheesman E. H., Maheu L. J., Hall K. M. Studies of the retention mechanism of the brain perfusion imaging agent 99mTc-bicisate (99mTc-ECD).
J. Cereb. Blood Flow Metab.
,
14
:
S4
-S11,  
1994
.
23
Esik O., Emri M., Csornai M., Kasler M., Godeny M., Tron L. Radiation myelopathy with partial functional recovery: PET evidence of long-term increased metabolic activity of the spinal cord.
J. Neurol. Sci.
,
163
:
39
-43,  
1999
.
24
Laughlin K. M., Evans S. M., Jenkins W. T., Tracy M., Chan C. Y., Lord E. M., Koch C. J. Biodistribution of the nitroimidazole EF5 (2-[2-nitro-1H-imidazol-1-yl]-N-(2,2,3,3,3-pentafluoropropyl) acetamide) in mice bearing subcutaneous EMT6 tumors.
J. Pharmacol. Exp. Ther.
,
277
:
1049
-1057,  
1996
.
25
Parliament M. B., Chapman J. D., Urtasun R. C., McEwan A. J., Golberg L., Mercer J. R., Mannan R. H., Wiebe L. I. Non-invasive assessment of human tumour hypoxia with 123I-iodoazomycin arabinoside: preliminary report of a clinical study.
Br. J. Cancer
,
65
:
90
-95,  
1992
.
26
Chapman J. D., Coia L. R., Stobbe C. C., Engelhardt E. L., Fenning M. C., Schneider R. F. Prediction of tumour hypoxia and radioresistance with nuclear medicine markers.
Br. J. Cancer
,
27 (Suppl.)
:
S204
-S208,  
1996
.
27
Lythgoe M. F., Williams S. R., Wiebe L. I., McEwan A. J. B., Gordon I. Autoradiographic imaging of cerebral ischaemia using a combination of blood flow and hypoxic markers in an animal model.
Eur. J. Nucl. Med.
,
24
:
16
-20,  
1996
.
28
Keyeux A., Ochrymowicz-Bemelmans D. Late response of the cerebral circulation to X-irradiation of the brain in the rat..
Late Biological Effects of Ionizing Radiation
,
Vol. 2
:
251
-260, IAEA Vienna  
1978
.
29
Keyeux A., Dunjic A., Royer E., Jovanovic D., Van de Merckt J. Late functional and circulatory changes in rats after local irradiation.
Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med.
,
20
:
7
-25,  
1971
.
30
Reinhold H. S., Hopewell J. W. Late changes in the architecture of blood vessels of the rat brain after irradiation.
Br. J. Radiol.
,
53
:
693
-696,  
1980
.
31
Breier G., Albrecht U., Sterrer S., Risau W. Expression of vascular endothelial growth factor during embryonic angiogenesis and endothelial cell differentiation.
Development (Camb.)
,
114
:
521
-532,  
1992
.
32
Millauer B., Wizigmann-Voos S., Schnurch H., Martinez R., Moller N. P., Risau W., Ullrich A. High affinity VEGF binding and developmental expression suggest Flk-1 as a major regulator of vasculogenesis and angiogenesis.
Cell
,
72
:
835
-846,  
1993
.
33
Minchenko A., Bauer T., Salceda S., Caro J. Hypoxic stimulation of vascular endothelial growth factor expression in vitro and in vivo.
Lab. Investig.
,
71
:
374
-379,  
1994
.
34
Senger D. R., Galli S. J., Dvorak A. M., Perruzzi C. A., Harvey V. S., Dvorak H. F. Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid.
Science (Washington DC)
,
219
:
983
-985,  
1983
.
35
Berkman R. A., Merrill M. J., Reinhold W. C., Monacci W. T., Saxena A., Clark W. C., Robertson J. T., Ali I. U., Oldfield E. H. Expression of the vascular permeability factor/vascular endothelial growth factor gene in central nervous system neoplasms.
J. Clin. Investig.
,
91
:
153
-159,  
1993
.
36
Murata T., Nakagawa K., Khalil A., Ishibashi T., Inomata H., Sueishi K. The relation between expression of vascular endothelial growth factor and breakdown of the blood-retinal barrier in diabetic rat retinas.
Lab. Investig.
,
74
:
819
-825,  
1996
.
37
Wang W., Merrill M. J., Borchardt R. T. Vascular endothelial growth factor affects permeability of brain microvessel endothelial cells in vitro.
Am. J. Physiol.
,
271
:
C1973
-C1980,  
1996
.
38
Roberts W. G., Palade G. E. Increased microvascular permeability and endothelial fenestration induced by vascular endothelial growth factor.
J. Cell Sci.
,
108
:
2369
-2379,  
1995
.
39
Wang W., Dentler W. L., Borchardt R. T. VEGF increases BMEC monolayer permeability by affecting occludin expression and tight junction assembly.
Am. J. Physiol. Heart Circ. Physiol.
,
280
:
434
-440,  
2001
.
40
Plate K. H., Beck H., Danner S., Allegrini P. R., Wiessner C. Cell type specific upregulation of vascular endothelial growth factor in an MCA-occlusion model of cerebral infarct.
J. Neuropathol. Exp. Neurol.
,
58
:
654
-666,  
1999
.
41
Hayashi T., Abe K., Suzuki H., Itoyama Y. Rapid induction of vascular endothelial growth factor gene expression after transient middle cerebral artery occlusion in rats.
Stroke
,
28
:
2039
-2044,  
1997
.
42
Lennmyr F., Ata K. A., Funa K., Olsson Y., Terent A. Expression of vascular endothelial growth factor (VEGF) and its receptors (Flt-1 and Flk-1) following permanent and transient occlusion of the middle cerebral artery in the rat.
J. Neuropathol. Exp. Neurol.
,
57
:
874
-882,  
1998
.
43
Proescholdt M. A., Heiss J. D., Walbridge S., Muhlhauser J., Capogrossi M. C., Oldfield E. H., Merrill M. J. Vascular endothelial growth factor (VEGF) modulates vascular permeability and inflammation in rat brain.
J. Neuropathol. Exp. Neurol.
,
58
:
613
-627,  
1999
.
44
Fitch M. T., Doller C., Combs C. K., Landreth G. E., Silver J. Cellular and molecular mechanisms of glial scarring and progressive cavitation: in vivo and in vitro analysis of inflammation-induced secondary injury after CNS trauma.
J. Neurosci.
,
19
:
8182
-8198,  
1999
.
45
Tofilon P. J., Fike J. R. The radioresponse of the central nervous system: a dynamic process.
Radiat. Res.
,
153
:
357
-370,  
2000
.