Cranial irradiation can result in numerous clinical sequelae, which contribute to the morbidity and mortality of patients. The pathogenesis of these adverse effects remains elusive, but a number of studies support a significant role for neuroinflammation. To establish a firm foundation for future mechanistic investigations, a major goal of the current study was to characterize the neuroinflammatory response of the C57BL/6 mouse brain over a wide range of doses and times after single-dose cranial irradiation. The doses used in this study are consistent with those used by other investigators (8
The early and late neuroinflammatory responses to cranial irradiation seen in our study are consistent with previous studies using mice and other species. Cranial irradiation of the C57BL/6 mouse resulted in dose-dependent, acute increases in mRNA levels of multiple proinflammatory mediators and markers of activation, as has been reported previously in different mouse strains (10
). Although no formal behavioral testing was conducted, no overt changes in behavior, grooming or balance were noted in our irradiated animals at any time, a result consistent with previous studies (8
). Indeed, the only grossly apparent difference between animals cranially irradiated with 25 and 35 Gy and controls, which became statistically significant at 2 months postirradiation, was the failure to demonstrate age-appropriate weight gain. Similar findings have been reported in cranially irradiated rats after doses of 25 Gy at 6 months (9
In the current study, some of the mice cranially irradiated with doses ≥25 Gy developed overgrown teeth, starting approximately 3 months postirradiation, that interfered with eating. As a result, animals were examined every 7 to 10 days, their teeth were clipped if necessary, and their food was placed at ground level for easier access. Based on dosimetry, the delivered dose was very localized, with essentially no total-body dose. We speculate, therefore, that the decreased weight seen at the two highest doses was partially due to the radiosensitivity of the salivary glands, which is well documented in mice (28
). Some salivary gland dysfunction was probably present after all of the doses used in this study; recovery of salivary function and some glandular sparing most likely explains the relatively normal weight gain seen at the two lower doses (29
In addition to demonstrating early and late neuroinflammatory changes seen in previous work, the current study did elucidate some important differences and novel findings. Unlike previous studies using C3H mice (10
), cranially irradiated C57BL/6 mice did not appear to suffer from increased mortality due to brain injury. There was no mention of abnormalities in dentition in other studies, so it is unclear whether this played a role in the mortality observed by others or whether the reduced mortality in our study reflects a potential strain difference in brain radiosensitivity. Alternatively, the dose range used may have been too low to elicit a lethal response. For example, the mice in our study did not show any histological evidence of radiation necrosis, consistent with previous reports (8
). In contrast, Jost et al.
60 Gy delivered to the left hemisphere of BALB/c mice in 10 equal fractions was required to elicit radiation necrosis; lower doses resulted in no detectable radiation necrosis out to 10 months (32
Our study also demonstrated an acute infiltration of neutrophils into brain at 12 h postirradiation, a phenomenon that has been described in other tissues (33
). This finding is not completely surprising because previous work demonstrated increased leukocyte rolling and adhesion at 2 and 24 h postirradiation in pial vessels (13
). Although the specific mechanisms were not evaluated, the increased expression of TNFA and ICAM-1 observed during this time frame likely contributed to neutrophil infiltration (7
). Neutrophils possess NADPH oxidase and, upon stimulation, can release a burst of superoxide radicals, leading to a further increase in oxidative stress (36
). We found an increase in the mRNA of heme oxygenase-1 (HO-1) after cranial irradiation at 24 h. HO-1 is a microsomal enzyme present in endothelial cells, microglia, astrocytes and neurons that is upregulated in response to inflammation, tissue injury and/or oxidative stress (37
); however, it is unclear if this early upregulation of HO-1 is the result of inflammatory stimuli or increased oxidative stress resulting from radiation exposure and neutrophil infiltration.
One of the more interesting and novel findings in this study was the delayed increase in CNS T-cell and dendritic cell populations after cranial irradiation. The central nervous system has long been described as being protected from the peripheral immune system or “immunologically privileged,” but this is not the case. Leukocytes are known to infiltrate the normal CNS and are believed to be involved in immunological surveillance of the parenchyma (40
). Previous reports have described infiltrating T cells in response to radiation, but these were involved with perivascular cuffing and radiation necrosis (24
). In this study, it appears that radiation increased T-cell surveillance in the CNS and that this persisted for up to 12 months postirradiation. To the authors’ knowledge, this is also the first report of dendritic cells appearing in the brain after cranial irradiation. Based on the times analyzed, it is unclear if the appearance of one cell type preceded another or if they appeared in response to the same stimuli.
Both T cells and dendritic cells showed a predilection for white matter. In addition, some CD11c-positive cells were found to be positive for MHC II, suggesting that these cells were mature dendritic cells and were capable of presenting antigen. T cells also appeared to be in proximity to dendritic cells (data not shown), suggesting that they may be interacting with one another. Interestingly, the appearance of T cells and dendritic cells in white matter and their proximity to each other have been reported previously in C57BL/6 mice as part of normal aging (45
). Further work needs to be performed to better characterize and elucidate the functions of specific subtypes of T cells and dendritic cells, in both normal and irradiated brain.
Cranial irradiation also caused a delayed, dose-dependent increase in the number of MHC II-positive cells that persisted out to 12 months. Similar findings have been reported in rat brain after 20 Gy (9
). The distribution of these cells in the brain was more homogeneous than that of the dendritic cells or T cells and did not show an obvious predilection for white matter. Consistent with upregulation of MHC II, these cells appeared to have an activated microglial morphology. Activated microglia are known to produce a number of inflammatory mediators, recruitment factors and proteases as well as reactive oxygen and nitrogen intermediates (46
). At 1 month, decreases in SOD2 and GSTP1 mRNA were noted (Supplementary Table 1
), and at 6 months, HO-1 mRNA levels were increased. These changes may be related to the persistent oxidative stress after radiation exposure that has been reported in the CNS by multiple investigators (48
). The activated microglia, along with dendritic cells and T cells, indicate that radiation induces a chronic neuroinflammatory environment, a hypothesis that is further supported by late increases in the mRNA levels for TNFA, MHC II, GFAP and CCL2.
Our study demonstrated that cranial irradiation induced the recruitment of T cells in the C57BL/6 mouse brain. MHC II-positive and CD11c-positive cells are not typically found in the young mouse CNS parenchyma under normal conditions; whether these myeloid cells arose from endogenous microglia or were recruited from the periphery after irradiation remains to be established. Evidence that CCL2, a known recruitment factor for myeloid-derived cells, is upregulated at 6 months suggests that it may play a role in recruiting cells to the irradiated brain. Indeed, 10 Gy of whole-body irradiation has been shown to recruit CD11c-positive cells from the periphery (50
). Future studies are planned to better answer this question of cell origin in our model using bone marrow chimeras.
Finally, in the context of clinical practice, there is speculation that the accumulation of immune cells may be beneficial in the treatment of primary brain tumors. Gliomas represent 32% of all primary brain and CNS tumors, with glioblastoma multiforme (GBM) accounting for greater than 50% of all gliomas (51
). GBM is the most aggressive type of glioma and carries an unfavorable prognosis, with a 1-year survival rate of 33% and less than 5%survival at 5 years (51
); with standard of care therapeutic intervention, the mean survival is 14.6 months (52
). As a result, efforts are under way to develop newer combination therapies that increase survival. One such approach is to combine immunotherapy with radiotherapy (53
). Two of the more popular immunotherapies are adoptive T-cell transfer and dendritic cell vaccines, where both sets of immune cells are modulated ex vivo
and returned to the patient (56
). Common hurdles for both approaches are overcoming immunosuppressive factors and immune avoidance of the CNS and CNS tumors to facilitate T-cell stimulation and cell killing as well as enhanced delivery of effector cells to the tumor site (56
). Importantly, the glial activation, endothelial activation and immune cell recruitment observed in this study appeared to be limited to areas that were exposed to the radiation beam, as demonstrated by the rostrocaudal distribution of MHC II+ cells. Therefore, this observation of radiation-targeted immune activation suggests that combination radiotherapy and immunotherapy could yield improved therapeutic outcomes. Indeed, a synergistic effect of the two modalities was recently demonstrated in a rat glioma model (58
In summary, our study demonstrates that cranial irradiation results in a bi- or multiphasic inflammatory response with a delayed infiltration of immune cells in the C57BL/6 mouse brain. In this model, cranial irradiation induced an acute recruitment of neutrophils and caused rapid endothelial and glial activation. This response was followed by a delayed infiltration of T cells beginning at 1 month postirradiation that was accompanied by the appearance of MHC II-positive dendritic cells. These cells remained in the brain for months, indicating that cranial irradiation leads to persistent neuroinflammatory changes in the C57BL/6 mouse brain. Further studies will be required to determine whether such alterations in the CNS microenvironment affect brain function or responses to other challenges, including age-associated changes.