Several studies have investigated the effects of radiation on the BBB or BTB, all reporting different results concerning permeability of brain barriers (49–51). Additional disparities are observed between reports owing to the non-uniform, clinically dissimilar dosing schemes. In this study we validate a new experimental design using the commercially available XenX Small Animal Irradiator and observed increased BBB permeability to TxRed 24hrs following a total dose of 12 Gy in immune competent animals only. Moreover, we also saw increased permeability of the BTB following low to moderate doses of radiation at 8 and 24hrs following radiation treatment.
In this work, first we validated our experimental design through small field radiation dosimetry using a combined ionization chamber and EBT3 Gafchromic® film approach. A similar approach using an equivalent radiation system has been used previously (52,53). Multiple groups have used dose rate measurements in solid water phantoms, cross calibrated with EBT3 films to gauge doses delivered for a particular experimental setup (54). Herein the dose rate for our small animal irradiator (SAI) at isocenter and an open field was determined to be 3.62 Gy/min, consistent with dose rates for similar field sizes (52). The irradiated field demonstrated quality beam uniformity (Fig. 2) in comparison with our intended field size and had a penumbra, where dose deposition falls from 80% of the max dose to 20% of the max dose, measuring 0.850 mm. Measurement and outcomes of beam uniformity and field penumbra for our experimental design are comparable, but vary slightly from others reporting a beam penumbra of 0.40–0.41 mm (55) using a 10 × 10mm2 field. While the beam penumbra is critical in small scale irradiation methodology, the intent of this work was to study the effect of radiation on tumors in a large treatment field consisting of half of the brain. For this purpose, a beam penumbra of < 1 mm would not deliver substantial dose to the region outside the intended field, nor would it prevent the intended field from receiving a significantly lower dose.
To translate from a dosimetric evaluation of our SAI and its beam characteristics, we transitioned to an in-vivo system. Using naïve female FVB mice and immunostaining, we were able to histologically verify successful irradiation of a brain hemisphere by increased γH2AX signal in the treated hemisphere (Fig. 3C). The use of anti-γH2AX staining to ascertain radiation damage, specifically double stranded DNA breaks, and field sizes in in-vivo systems has been established (52,56,57).
In order to understand the effects of WBRT on the normal brain and brain tumor vasculature, we modeled clinical dosing patterns to treat and ablate brain metastases. Patients are commonly prescribed a total dose of 30 Gy over 10 fractions (58,59). When fractionation schemes are used, it is critical to understand their translational relevance. One group (60) studied the effects of fractionated radiotherapy on the BBB and BTB in rats. While the dosimetry was well executed, the doses and fractionation patterns do not appear to match what is typically used in patients in the clinic. In a similar study (31), mice were treated with a single fraction of 10 Gy. Interestingly, Zarghami et al. (56) limited doses to single fractions, but incorporated the use of a BED equation to demonstrate equivalence to clinical dosing parameters. Of note, changes in fractionation have shown little impact on tumor progression and survival (59).
However, when examining the effects of a treatment on the blood brain barrier, it is important to follow clinical parameters and understand the intent of the treatments. Our experiments were poised to examine the events following a radiation treatment intended to treat brain tumors. Doses outside of what are typically used in patients are not necessarily as translationally plausible as studies using methods employed in the clinic. Our findings are presented at low and moderate doses, but were given in the same 3 Gy fractions that would be continued to 30 Gy in the clinic.
In non-tumor bearing, healthy female Nu/Nu mice, the BBB was unaffected by radiation therapy at doses from 0-12Gy in fractions of 3 Gy at 24hrs following treatment (Fig. 4A). Contrary to our results, Wilson et al (30) demonstrated increased normal BBB permeability to a 4.4 kDa FITC dextran at 24 and 48hrs following radiation. However, this result was following a single exposure to a relatively large, 20 Gy dose of radiation. Using the BED equation, this equates to an effective dose that is greater than 1.5 times that of a total dose of 30 Gy over 10 fractions (61). Another study using a single dose of 20 Gy that used various sized FITC dextran molecules observed increased permeability peaking at 24hrs post-treatment. However, they observed no increases in normal BBB permeability following a dose of 5 Gy, which is much closer to the single fraction dose we used in our work (37). The differences in reported measurement of BBB permeability alterations following radiation therapy can be partially attributed to the large heterogeneity in the way the dose was delivered, i.e. high dose vs low dose or single vs multiple fractions.
While our results using athymic nude mice may conflict with reported data, experiments with mice bearing an intact immune system had a different outcome. When immune competent female FVB mice were used in the same experiment, we observed a significant increase in normal BBB permeability to TxRed 24hrs following a dose of 12 Gy, as well as an increased, albeit not significant, permeability change 24hrs following a dose of 6 Gy (Fig. 4C). It should be noted that in the previously discussed experiments, immune-competent rodent models were used (30,37). These results suggest an active role of the peripheral and CNS immune system in BBB regulation following radiation therapy. Increased cytokine expression has been observed following treatment with radiation (62–64). Specifically, TNFα, IL1β, and IL6 have increased expression, similar to acute periods after neuro-immunological insults (65,66). Additionally, at a cerebral blood flow rate of 2 mL/min/g (67), immune cells traversing the cerebrovascular network will be exposed to a substantial dose of radiation, more than likely perturbing an inflammatory response. The damage associated molecular patterns released and innate immune cell cytokine production following radiation therapy could potentially amplify this immune response (10,68,69). All of the underlying inflammatory events following radiation treatments may result in a potential mechanism for BBB disruption in immune competent subjects.
Lastly we set out to determine the effects of WBRT on the vascular system within metastatic brain tumors. Our data indicated increased BTB permeability at both 8 and 24hrs following treatment with 6 Gy of radiation in 2 fractions, while after 24hrs we saw increased BTB permeability following a dose of 12 Gy in 4 fractions (Fig. 5). This data is consistent with increased Ktrans values (BBB permeability measured clinically) seen in quantitative DCE MRI in irradiated tumors at 24hrs post-irradiation (70). Broad beam radiotherapy also displayed increased BTB permeability in treated lesions (71). Tumor vasculature response has also been studied clinically. In 30 patients and 64 total lesions receiving WBRT or SRS, treatment with radiation increased permeability in initially low leaky tumors (72). However, in tumors that were already highly permeable, there were no significant increases in permeability. In opposition to what we have observed in this study, there have been observations of no permeability changes measured by MRI gadolinium enhancement (51), though a dose of 20 Gy over two fractions was given. While this is different from our study in terms of single fraction dose and fraction number, the BED is similar to that of a completed 30 Gy in ten fractions. For a better visualization of how our results align with concluded studies, pertinent data available in the literature for both preclinical and clinical experiments are organized in table 1.