Many recent studies have focused on damage to the endothelial cells as a key target (15, 60). The importance of the vasculature was illustrated in boron-neutron capture therapy (BNCT) studies. Using capture agents to selectively irradiate the spinal cord microvasculature, histopathologic changes observed in rats that developed myelopathy after BNCT were virtually identical to those observed in animals treated with X-rays or neutrons only (61). These studies were confirmed by comparison of the surviving fraction of clonogenic oligodendrocyte progenitor cells after treatments with capture agents that did or did not cross the blood spinal cord barrier, or with treatment by thermal neutron beam only. The surviving fractions of clonogenic progenitors were significantly higher when the radiation dose was primarily delivered in the vascular endothelium, despite the fact that all treatments resulted in an equal incidence of white matter necrosis (51).
Consistent with this notion is the late disruption of BBB as a consistent finding that precedes gross white-matter damage in the CNS after XRT (62–64). We shall now focus on the disruption of the BBB and the associated micro-environmental changes that may lead to tissue damage.
Using the electron microscope to evaluate changes in endothelial cells in rat spinal cord, Stewart et al. noted a 30% reduction of microvessel endothelial density in the white matter, observed as early as three months after a single dose of 25 Gy (63). This observation was associated with focal disruption of the BSCB in white matter. It is unknown whether early endothelial apoptosis contributes to late microvessel density changes and BBB disruption. Failure of repair of early barrier disruption may also evolve into persistent barrier incompetence.
HYPOXIA AND VEGF
Hypoxia develops where oxygen supply from the vasculature is compromised because of deficient vascularization or local microcirculation disturbances, such as in ischemic or traumatic injury. 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. Additionally, hypoxia is a crucial stimulus for vascular endothelial growth factor (VEGF)––also known as vascular permeability factor––which is known to mediate increased permeability in a wide range of tissues including the CNS (65). In the neonatal rat spinal cord, induced VEGF expression was observed within days and persisted for two weeks after a very high dose of XRT (55 Gy). This observation was followed by an increase in vascular density at four to five weeks after XRT (66). In the adult rat myelopathy model, where rats develop paralysis associated with necrosis of the white matter within twenty weeks after single doses of 20 to 25 Gy, a steep increase in the number of VEGF-expressing cells was observed beginning at sixteen weeks post-treatment. The increase in the number of VEGF-expressing cells also demonstrated a dose-dependent response above 17 Gy. The majority of these cells were astrocytes (67).
In a subsequent study (10), hypoxia in the irradiated rat spinal cord was assessed using two 2-nitroimidazole markers, [125I]- iodoazomycin arabinoside (IAZA) and 2-(2-nitro-1H-imidazol-1-l)- N-(2,2,3,3,3-pentafluoropropyl) acetamide (EF5), measured in the rat spinal cord using gamma-ray scintillation counting and immunohistochemistry, respectively. BSCB permeability was assessed using immunohistochemistry with an albumin-specific antibody and gamma-ray scintillation counting of [99mTc]-diethylenetriamine pentaacetic acid (DTPA). A dose-dependent increase in albumin staining and [99mTc]-DTPA activity beginning at sixteen weeks was observed, consistent with barrier breakdown. A similar dose-dependent increase in white matter astrocytes that showed immunoreactivity and in situ hybridization signals for VEGF was also observed. Irradiated rat spinal cord showed a dose- (17–22 Gy) and time-dependent (16–20 weeks after 22 Gy) increase in accumulated [125I]-IAZA compared to [125I]-IAZA accumulation non-irradiated controls. A similar pattern of dose- and timedependent EF5 immunoreactivity was also observed in white matter. Areas of EF5 expression and VEGF in situ hybridization signals co-localize with areas of albumin immunoreactivity. These results provided evidence for a dose-dependent temporal and spatial association of hypoxia, increased VEGF expression, and radiation- induced BSCB dysfunction.
To examine the functional consequences of altered VEGF expression, the response of VEGF-lacZ knock-in transgenic mice with increased or decreased functional VEGF expression to spinal cord XRT was assessed (12). Following XRT to the thoracolumbar spinal cord, transgenic mice with reduced VEGF showed protection and had a longer median time to development of weakness and paralysis compared to wild type mice and transgenic mice with increased VEGF. These results suggest a causal role for VEGF in the development of radiation myelopathy, and provide clear targets for intervention.
HYPOXIA AND OTHER HIF1-TARGET GENES
In the rat radiation myelopathy model, the number of astrocytes expressing hypoxia-inducible factor-1
(HIF1), VEGF, and glucose-transporter-1 (Glut-1) increased with increasing doses of XRT above 17 Gy, and with increasing time after sixteen weeks following 20 Gy treatment. There was also spatial co-expression of HIF1, VEGF and Glut-1 in regions of the spinal cord with evidence of hypoxia and BSCB disruption (12).
Reactive oxygen species are implicated in a number of degenerative and injurious processes in the CNS (68). They may play a role in CNS radiation injury (30). Various mechanisms may lead to the generation of free radicals under hypoxic conditions (69, 70). HO-1, a protein of oxidative stress, is induced by hypoxia through HIF1 (71). Upregulation of HO-1 in the rat spinal cord at 5 and 6 months after 26 Gy has been reported as evidence for oxidative stress (2).
Increased HIF1 may also upregulate the HIF1-target genes lactate dehydrogenase and other glycolytic enzymes such as phosphofructokinase, and aldolase-A. LDH induction is potentially harmful because it may lead to increased lactate production (72, 73). Whether this contributes to the altered microenvironment in the CNS under hypoxia remains uncertain.
ENDOTHELIAL TIGHT JUNCTION PROTEINS, TIGHT JUNCTION INTEGRITY, AND BBB DISRUPTION
The underlying mechanisms of VEGF-mediated increase in vascular permeability are unclear. There is evidence that VEGF-mediated vascular permeability changes do not require receptor binding (74). In radiation-induced late BBB disruption, VEGF upregulation was not associated with evidence for increased or even detectable VEGF receptor expression (12).
The integrity of the BBB is dependent on tight junctions, and possibly adherens junctions, between endothelial cells (75). VEGF increases the permeability of the brain endothelial cell monolayer and alters the expression and distribution of occludin and zonula occludens-1 (76). Incubation of umbilical vein endothelial cells with VEGF increased their permeability and decreased occludin expression (77). There is also evidence that hypoxia-induced increases in permeability of brain endothelial cells involve VEGFmediated changes in expression of zonula occludens-1 (78).
In a morphometric study using the electron microscope, no disruption or expansion of microvessel tight junction contacts was detected in the irradiated rat spinal cord (63). A different study demonstrated no apparent change in the distribution or amount of immunoreactivity of the tight junction proteins occludin and zonula occludens-1 in rat spinal cord after myelopathic radiation doses (11).
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