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British Journal of Radiology (2004) 77, 488-492
© 2004 British Institute of Radiology
doi: 10.1259/bjr/15169876
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Full Paper

Experimental evidence to support the hypothesis that damage to vascular endothelium plays the primary role in the development of late radiation-induced CNS injury

N Lyubimova, PhD 1 and J W Hopewell, DSc 2

1 Institute of Theoretical and Experimental Biophysics, Russian Academy of Science, Pushchino, Moscow Region, 142292, Russia and 2 Department of Clinical Oncology, Oxford Radcliffe Hospitals NHS Trust, The Churchill Hospital, Oxford OX3 7LJ, UK


   Abstract
Top
 Abstract
Introduction
 Material and methods
 Results and discussion
 References
 
Experimental evidence has been obtained to support the view that late necrosis in the brain, after irradiation, is a consequence of damage to the vascular system. Rats were locally irradiated to the brain with a single dose of 25 Gy of X-rays and their response was compared with rats given the same treatment after administration of the radioprotector, Gammaphos. Time-dependent changes in endothelial cell number were determined for up to 65 weeks after irradiation. The complex pattern of changes in endothelial cell number, seen after irradiation alone, was not found in animals receiving Gammaphos prior to irradiation. The initial marked loss of endothelial cells, seen after 24 h in unprotected animals, was effectively prevented by the pre-administration of Gammaphos. The subsequent slow decline in endothelial cell density in Gammaphos treated animals was insufficient to induce an abortive attempt at endothelial cell re-population. This occurred between 26 and 52 weeks after irradiation in unprotected animals. By 65 weeks after irradiation <10% of animals receiving Gammaphos showed necrosis on histological evaluation. This compared with approximately 50% of the animals showing necrosis that had not received the radioprotector. Since the radioprotector is restricted to the vasculature of the brain these data indicate that endothelium is the primary target cell population, damage to which leads to the development of late radiation-induced necrosis in the brain.


   Introduction
Top
 Abstract
 Introduction
Material and methods
 Results and discussion
 References
 
Late effects of radiation can appear months, even years, after exposure. The main changes to occur include tumour induction, life shortening, pathological modification to blood vessels, tissue necrosis and fibrosis. These changes tend to develop after exposure to a specific range of radiation doses. This communication concentrates on the issue of late radiation-induced damage after acute radiation exposure of the brain and in particular, on the relationship between changes in endothelial cell density and the late development of pathological alterations in blood vessels and brain necrosis.

Despite an improved understanding of the molecular basis of radiation response, questions remain concerning the cell–cell interactions involved in the development of radiation-induced pathologies and in defining which cell populations are the primary targets responsible for the development of radiation damage. In the central nervous system (CNS) this remains an unresolved issue. It has been suggested [1] that late radiation effects in normal tissues are the consequence of damage to various critical cell systems. The tissue and the radiation dose-range will determine the critical target cell population. In the brain, two cell populations, glial cells and vascular endothelial cells, are potentially target cells. The differential irradiation [2] or selective radioprotection of either endothelial, as in the present study, or of the glial cell population could help to elucidate their relative importance in the development of late radiation injury to this tissue.

An earlier study provided circumstantial evidence for a critical role for endothelial cells in the development of brain necrosis. Endothelial cell numbers in the rat brain were monitored after irradiation using a fluorescent histochemical technique [3]. This showed that within 1 day of irradiation there was a fall in endothelial cell number to about 85% of that of age-matched controls. In view of the slow turnover of these cells [4], this is most likely to be explained by the apoptosis of a sensitive sub-population of endothelial cells [3, 5]. (The existence of this separate sub-population sensitive to apoptosis, around 15% of all endothelial cells, suggests that these endothelial cells may have a significantly different role from other endothelial cells [3]. This possibility requires further investigation.) The rapid loss of cells within a day was followed by a further slow loss of endothelial cells over several months. When cell numbers became depleted to about 65% of normal, an attempt at re-population was observed. However, this was transient, and a second decline in endothelial cell density then occurred, shortly before the onset of brain necrosis.

Recently, a related study was carried out on mice using the radioprotector, Hoechst 33342. This was administered shortly prior to irradiation [5]. This radioprotector rapidly localizes in endothelial cells but is not present elsewhere in brain at the time of radiation exposure. It was found that pre-treatment with Hoechst 33342 substantially protected endothelial cells against the initial loss (24 h) after low doses and that this was associated with a dose modification factor of ~3.0. In so far as endothelial cells are the likely critical target for radiation damage leading to late necrosis, it seemed possible that late brain necrosis might also be ameliorated by the selective radioprotection of endothelial cells. However, the study with Hoechst 33342 was limited to the evaluation of the initial rapid (apoptotic) loss of endothelial cells; it did not address the more important question as to whether the initial protection observed extends to the longer-term loss of endothelial cells and if the protection of endothelial cells modifies the incidence of late brain necrosis.

The present paper addresses these questions directly, testing the hypothesis that endothelial cell loss is critically responsible for late radiation-induced necrosis in the brain. Data are presented on the time-related changes in endothelial cell density, on the incidence of both gross vascular changes and necrosis in the brain of Wistar rats receiving the radioprotector Gammaphos (manufactured by the Institute of Biophysics, Ministry of Health, Russia, but chemically identical to WR-2721) before local irradiation with a single dose of 25 Gy of X-rays. This dose is known to be the minimum required to produce selective late necrosis of white matter in some regions of the rat brain but without deaths or detectable neurological changes [6]. This radioprotector was chosen because it does not cross the blood–brain barrier (BBB) [7], thereby restricting radioprotection to endothelial cells, leaving the radiosensitivity of glial cells unchanged. The timing of the maximal blood concentration of WR-2721 after intraperitoneal (i.p.) administration is known from experiments with 35S-labelled WR-2721 [8]. These data were used to ensure that, for the present experiments, a maximal concentration of protector was available in the CNS vasculature during irradiation.


   Material and methods
Top
 Abstract
 Introduction
 Material and methods
Results and discussion
 References
 
8-week old male Wistar rats weighing 180–200 g were used. Prior to irradiation rats were anaesthetized with an i.p. injection of sodium pentobarbital (60 mg kg–1). The brain was then locally irradiated, bilaterally, with a single dose of 25 Gy of X-rays (200 kV at 3 Gy min–1, 1 mm Cu and 1 mm Al added filtration, focus–skin distance 22.8 cm). Protection of the endothelial cells was achieved by the injection of Gammaphos, 350 mg kg–1, 3 min before radiation exposure. Results from these animals are compared with those from concurrently irradiated animals receiving 25 Gy alone. The results from those animals have been published previously [3].

Changes in endothelial cell number were determined at 24 h after irradiation and then at intervals of 2, 4, 9, 26, 39, 45, 52 and 65 weeks after exposure for both Gammaphos and non-Gammaphos pre-treated animals and in age matched, unirradiated control, animals. To facilitate the counting of endothelial cells in histological sections a fluorescent marker was produced in endothelial cells [3]. Animals were first injected with 100 mg kg–1 of iproniazid (i.p.), an inhibitor of monoamine oxidase that normally controls the levels of biogenic amines crossing the BBB. Approximately 6 h later and just before the animals were killed, according to the local animal care committee protocols, an injection of 100 mg kg–1 of the catecholamine precursor L-DOPA was given i.p. Due to the BBB, this combination of pharmacological agents resulted in an accumulation of biogenic amines in endothelial cells, namely of catecholamines (CA). While under sodium pentobarbital anaesthesia, rats were killed by decapitation and samples of brain taken from between the posterior margin of the olfactory bulb and the optic chiasma. These samples were immediately frozen in liquid nitrogen. The brain samples were then lyophilized at –20°C under vacuum to prevent CA diffusion from endothelial cells. The reaction between CA and para-formaldehyde gas (70% humidity, 80°C) was used to generate fluorophores, as an endothelial cell marker.

Samples for histological evaluation were embedded, under vacuum, in paraffin wax. Coronal sections of the brain, 15 µm thick, at the level of posterior–anterior junction with the optic chiasma were used in these evaluations using a fluorescence microscope (excitation 410 nm, emission 480 nm). The effect of irradiation on endothelial cell density was assessed in grey (cortex), white (corpus callosum) and mixed grey-white (septum) matter regions for up to 65 weeks after irradiation. In all three morphological regions, up to 20 fields were scored (each 0.044 mm2), corresponding to a total count of about 700 endothelial cells per histological section of rat brain, as described previously [3]. Additional, adjacent, 15 µm thick sections from the same material were cut and stained with haematoxylin-eosin (H&E) and Nissl method with cresyl violet. These sections were used to assess the presence or absence of gross vascular changes or tissue necrosis according to criteria used previously [6, 9].

In order to assess changes in blood vessel density an additional group of animals was irradiated with 25 Gy, with and without the prior administration of Gammaphos, as described above. At intervals of 17, 26, 39, 52 and 65 weeks after irradiation these animals, and age-matched unirradiated control animals were anaesthetized with sodium pentobarbital and the vasculature perfused, at constant pressure, by the left ventricular injection of a warm suspension of India ink in 4% gelatine. Death ensued within a minute of the start of the infusion. The brain was then removed and fixed in 10% buffered formal saline. Sections were cut at 15 µm and assessed by computer image analysis from light microscope images captured by a TV-camera, as described previously [3]. This automated analysis of the capillary network, filled by India ink, provided an estimate of the average number of vessel cross sections per field with accuracy of 5%. To avoid mistakes related to the use of small areas of tissue, only two morphological regions, the cortex and septum were scored in 20 fields (each 0.16 mm2).

For the various evaluations at least five animals per group were used and results have been expressed as the mean±standard error (SE). Changes in cell and vessel density between different morphological regions have been evaluated by the Student's t-test.


   Results and discussion
Top
 Abstract
 Introduction
 Material and methods
 Results and discussion
References
 
Time-related effects of irradiation on the endothelial cell density, with respect to age-matched controls, in different morphological areas, are given in the Table 1Go. Significant differences in the response of the endothelium to radiation in the three brain regions were not seen except at 26 and 65 weeks after radiation. Because of the similarities in response in the different areas, the data over all three brain regions have been combined. The time-dependent average changes in endothelial cell density, relative to controls, after pre-treatment with Gammaphos and after 25 Gy of X-rays alone are shown in Figure 1Go.


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  		Table 1. Relative density of endothelial cells (irradiated/control) in different morphological regions of the rat brain after local irradiation with single doses of 25 Gy given alone or in combination with Gammaphos (GPh), injected intraperitoneally prior the irradiation. Results expressed as mean±standard error

 


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  		Figure 1. Time-related changes in endothelial cell number, relative to numbers in unirradiated control rats, after local brain irradiation with 25 Gy of X-rays. Animals received Gammaphos (•) before irradiation, or radiation alone (o). Error bars indicated±standard error.

 
After the initial reduction in endothelial cell density by 24 h, the changes in the endothelial cell population seen in animals receiving radiation only showed a classic pattern of response of a so-called "F" type [10] or "conditional renewal" [11] structure. After the initial loss of cells by apoptosis, the endothelial cell number declined slowly over a number of weeks at a rate comparable with calculated normal rate of endothelial cell turnover [4, 12], due to an effective lack of cell replacement by reproductively sterilized cells. Once feedback mechanisms were triggered, around 26 weeks after exposure at an endothelial cell density of approximately 65% of that of age-matched controls, there was an attempt at re-population. However, this wave of re-population was short-lived (probably as a consequence of an avalanche in cell death in radiation sterilized cells) and a rapid decline in the endothelial cell population ensued. Similar patterns of endothelial cell response have also been reported in the choroid plexus of the rat brain [13] and in other organs [14, 15].

The pattern of endothelial cell loss seen in the animals receiving Gammaphos differed markedly, as was the case in the studies in mice using the radioprotector Hoechst 33342 [5]. The early loss of cells, presumably by apoptosis, was prevented and the number of endothelial cells found 24 h after irradiation was not significantly different from that found in age-matched control animals. Although there was evidence for a subsequent slow loss of endothelial cells in the Gammaphos treated group, to around 85% of age-matched control values, this was clearly insufficient to provoke either the an attempt at re-population or the avalanche of cell death seen in animals receiving radiation only. From around 26 weeks after irradiation the endothelial cell density in the rats receiving Gammaphos remained almost constant at about 85% of age-matched control values.

The results of the micro-angiographic evaluation, to determine the average changes in blood vessel density in two brain regions, relative to age-matched controls, in locally irradiated animals both with and without pre-treatment with Gammaphos are shown in Figure 2Go. Slightly reduced blood vessel density was seen at 26 and 65 weeks after irradiation alone, which corresponded to the times of maximum endothelial depletion. However, neither radiation alone, nor in combination with the radioprotector, induced a major change in the blood vessel density in cortex and septum over the 65 weeks observation period. This result probably reflects the substantial ability of the remaining endothelial cells to adapt and maintain a covering of the blood vessel wall.



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  		Figure 2. Time-related changes in blood vessel density, relative to density in unirradiated control rats, after local brain irradiation with 25 Gy of X-rays. Animals receiving Gammaphos prior to exposure ({blacktriangleup}) or irradiation only ({Delta}). Error bars indicated±standard error.

 
Examination of adjacent histological sections, stained with H&E, from the groups of rats used for the counting of endothelial density at 39, 52 and 65 weeks after irradiation, clearly indicated a latency period of approximately 52 weeks before the first appearance of foci of necrosis in animals receiving irradiation alone. The data for both vessel damage (mainly teleangiectasia) and focal necrosis are presented in Figure 3Go. An increased incidence of vascular abnormalities was first noted at around 39 weeks after exposure in rats receiving radiation alone. Between 52 and 65 weeks after exposure, and at the same time as the avalanche of endothelial cell loss in the irradiation-only animals, the incidence of both vascular abnormality and necrosis increased markedly. In contrast, in the rats receiving pre-treatment with Gammaphos, both vascular abnormalities and necrosis showed a lower incidence throughout the observation period. By 65 weeks after irradiation less then 10% of these animals showed vascular abnormalities and brain necrosis compared with over 75% and 55%, respectively, in rats receiving radiation alone.



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  		Figure 3. Time-related changes in the percentage incidence of both necrosis ({blacktriangleup}, {Delta}) and vascular abnormalities (•, o) in rat brain after irradiation with 25 Gy of X-rays. For both parameters closed symbols correspond to data from animals receiving Gammaphos prior to exposure; open symbols correspond to data for the group of animals receiving irradiation only. Error bars indicated±standard error.

 
It is interesting to note that vascular abnormalities and necrotic lesions were found predominantly in the white matter. Although differences in glucose metabolism and oxygen uptake have been reported between white and grey matter regions [16] and could play a role in their differential sensitivity to radiation, it is also well known that the blood vessel density is higher in grey matter compared with white matter.

In the present study the linear density (number of endothelial cells per 1 mm along the capillary wall) was also examined in grey and white matter using the same 15 µm sections used to score the endothelial cell density. In unirradiated control brains it was found that the separation of the endothelial cells was such to give approximately 24.4±0.5 endothelial cells per 1 mm capillary length in white matter, compared with 33.0±0.3 per 1 mm capillary length in grey matter. Such counts are necessarily biased, as pairs of widely separated endothelial cells are much less likely to lie within the section and thus capillaries with very widely separated endothelial cells will be excluded from the analysis. Nevertheless this approximate, 1.4 times lower linear density of the cells along the capillaries in white matter does suggest that vessels in this region may be less well buffered against the effects of cell loss than those in grey matter. The more widely separated cells in white matter capillaries would require greater flexibility if they were to maintain continuity after cell loss. Thus the data presented are consistent with the suggestion that the capillary structure, in terms of the linear density of endothelial cells along vessel walls, could be critical in determining the location at which necrosis develops.

In conclusion, the present experiments confirm the results of an earlier study [5], with Hoechst 33342, showing that protection against the radiation-induced loss of endothelial cells in brain vasculature can be achieved. The most probable causal sequence is endothelial cell loss, abnormal microvascular circulation, "nutritional" insufficiency and finally necrosis of brain parenchyma. Most importantly, the present study shows that radiation-protection of the endothelial cell population has a significant effect on the development of late radiation damage. Since the presence of the BBB meant that only endothelial cells can be protected by the pre-administration of Gammaphos it also provides good evidence that it is endothelial rather than glial cell damage that leads to the late brain necrosis after a single radiation exposure of 25 Gy.


   Footnotes
 
Address correspondence to Professor J W Hopewell. Back

Current address for Dr Lyubimova; Institute of Pathology, University of Bern, CH-3010 Bern, Switzerland. Back

Received for publication June 13, 2003. Revision received December 3, 2003. Accepted for publication December 11, 2003.


   References
Top
 Abstract
 Introduction
 Material and methods
 Results and discussion
 References
 

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