The CNS, it has been argued, has a limited repertoire of responses to injury. The pathophysiology of CNS radiation injury shares many similar damage pathways with ischemic, inflammatory, and demyelinating insults. Our own data and emerging data from the literature are consistent with a model where alterations of the microenvironment (e.g., endothelial dysfunction, BBB disruption, microglial activation, and inhibition of neurogenesis) contribute to the final insult. Thus, another approach is to direct efforts at blocking or modifying damage-associated changes in the microenvironment. In this paradigm of injury, an important potential neuroprotector that has a pleiotropic function is erythropoietin (EPO).
ERYTHROPOIETIN AS PLEIOTROPIC NEUROPROTECTOR
EPO has long been recognized for its central role in erythropoiesis. It is a glycoprotein produced by the kidney and is responsible for the proliferation, maturation, and differentiation of the precursors of the erythroid cells (102). Human recombinant EPO has been used for over a decade to treat anemia in uremic and cancer patients.
There is evidence that EPO may regulate neurogenesis. Mice lacking EPO receptors (EPOR) die at embryonic day 13.5 (E13.5). The fetal brains of EPOR–/– mice have reduced numbers of neural progenitor cells and extensive apoptosis (103). EPORs are expressed in the adult subventricular zone, and EPO infusion into the adult lateral ventricles results in decreased numbers of neural stem cells in the subventricular zone and an increase in newly generated cells migrating to the olfactory bulb (104). EPOR expression has been observed in neurons, astrocytes, and microglia (105).
The pleiotropic functions of EPO are now well recognized (106). Similar to its regulation in erythroid tissue, EPO within the CNS is inducible by hypoxia and is regulated by HIF1(107, 108). Many cellular effects of EPO in the CNS have been described. They include inhibition of apoptosis, anti-inflammatory and antioxidative effects, prevention of glutamate-induced toxicity and stimulation of angiogenesis (109, 110).
There is now an emerging body of data on the neuroprotective effect of EPO against a wide variety of CNS insults (109). EPO infusion into the lateral ventricles of rodents prevents ischemiainduced learning disability and rescues hippocampal neurons from ischemic damage (111). EPOR is abundantly expressed at brain capillaries, and systemically administered EPO is able to cross the BBB to enter the brain (112). Indeed, systemic administration of EPO protects the rodent brain against ischemia-induced injury, concussive brain injury, immune damage in experimental autoimmune encephalomyelitis, and kainate-induced neurotoxicities (112). EPO also attenuates secondary inflammation in a rat spinal cord contusion model (113).
We have recently assessed hippocampal-dependent learning and memory function in mice after XRT using an eight-arm radial maze. At two and four months, mice given 17 Gy to the whole cranium showed a decrease in the number of correct entries and an increase in time to complete all eight entries. Animals irradiated to 17 Gy and given intraperitoneal EPO one hour after XRT showed no impairment compared to controls. EPO given alone without XRT did not influence performance at the radial maze, and locomotor activities were not impaired in mice at two and four months after XRT with or without EPO using open field test (114). Although the underlying mechanisms of these effects of EPO remain to be investigated, these results suggest that the neuroprotective role of systemically administered EPO may be applied to radiation injury.
EPO has been used extensively over the last ten to fifteen years for the treatment of anemia in cancer patients. Hence, any neuroprotective effects observed in preclinical models can be readily translated to the clinic. Similar to any biologic interventions, the potential adverse effect of EPO on tumor control needs to be investigated (115, 116).
TARGETING HYPOXIA AND HIF1 TARGET GENES
Tissue oxygen conditions could be improved by increasing the amount of oxygen inspired. Despite some anecdotal reports of efficacy (117, 118), studies of hyperbaric oxygen in CNS radiation injury have not demonstrated a benefit (119). This may indicate a fundamental problem due to microvascular dysfunction; however, hyperbaric oxygen is normally used after tissue necrosis has developed to stimulate vasculogenesis. Alternatively, reoxygenation injury may paradoxically accelerate axonal injury and abrogate any potential benefits (120).
An alternative strategy is to direct intervention at hypoxiainduced genes or proteins, rather than at hypoxia or HIF1. In addition to EPO, augmenting the expression of genes such as HO- 1 and Glut-1 may be of value. Enhancement of HO-1 responses might confer protection in hypoxia-induced pulmonary hypertension (121) and protect hypoxic cardiomyocytes against reoxygenation injury (122).
Transgenic mice with decreased VEGF activity were protected against radiation-induced myelopathy (12), suggesting that the inhibition of VEGF may confer protection. Barrier disruption may also provide a window of access for small molecules administered systemically to inhibit VEGF. Many available inhibitors of VEGF block receptor activation (123, 124). However, permeability increases that are associated with VEGF might not require its binding to the VEGFR. VEGF induces phosphorylation of tight junction proteins occludin and zonula occluden 1 (125). Other strategies thus may include modulating the effects of VEGF on tight junction proteins. VEGF-mediated increases in vascular permeability are associated with endothelial nitric oxide synthase (eNOS) activity and release of nitric oxide (NO) (83). Inhibition of NOS activity might therefore be used to reduce vascular dysfunction.
XRT is associated with induction of inflammatory markers including cytokine expression (28, 31). An increase in cyclooxygenase-2 (COX-2) expression and COX-2-mediated prostanoid production was observed in the irradiated mouse brain (17, 31). COX-2 is one of two isoforms of the obligate enzyme in prostanoid synthesis and a principal target of non-steroidal anti-inflammatory drugs (NSAIDs). Inhibition of COX-2 attenuates prostanoid induction and cerebral edema in mice after XRT (17).
XRT-induced inhibition of neurogenesis accompanies inflammation in the microenvironment and activation of the microglia (13). Indomethacin (an NSAID agent) given daily after XRT decreases microglial activation and restores neurogenesis within the subgranular zone of the dentate gyrus (9). The administration of indomethacin or other NSAIDs normalizes vascular permeability in intracranial gliomas (126). Reducing the inflammatory status of endothelial cells––as measured by expression of chemokines and ICAM-1––may represent the underlying mechanism of neuroprotection. Indomethacin may also exert its neuroprotective effect through mechanisms related to NO neurotoxicity rather than through COX-2 inhibition (127).
Increased trafficking of neutrophils into the CNS has been described for a number of injury models. In rat brain, increases in leukocyte–endothelial cell interactions were seen twentyfour hours and also three weeks after XRT in association with decreased blood flow (128). In a rat brain window model, the increase in adherent leukocytes was associated with increase in BBB permeability (49). Enhanced leukocyte trafficking has largely been ascribed to the adhesion molecule ICAM-1. The expression of ICAM-1 is induced in mouse brain and rat spinal cord after XRT (11, 35, 36, 129); it thus follows that increased leukocyte trafficking into the CNS may exacerbate the inflammation and BBB disruption induced by XRT injury.
Using a monoclonal antibody or another antagonist to block the extracellular interactions of ICAM-1 at the vessel lumen, one might be able to interrupt pathways activated by the binding of leukocytes, fibrinogen, or other interactions with as yet unknown binding partners. Injection with an ICAM-1–specific monoclonal antibody reduces leukocyte adhesion and BBB disruption in the rat cranial window model (49). Following thoracic XRT, ICAM-1 and inflammation contribute to pulmonary fibrosis and injury (130), and expression of VCAM-I and ICAM-I in the irradiated mouse lung was decreased by manganese superoxide dismutase– plasmid/liposome gene therapy (131). These findings identify targeting adhesion molecules as a potential molecular intervention to reduce neuroinflammation