Aging masks detection of radiation-induced brain injury

Abstract

Fractionated partial or whole-brain irradiation (fWBI) is a widely used, effective treatment for primary and metastatic brain tumors, but it also produces radiation-induced brain injury, including cognitive impairment. Radiation-induced neural changes are particularly problematic for elderly brain tumor survivors who also experience age-dependent cognitive impairment. Accordingly, we investigated i] radiation-induced cognitive impairment, and ii] potential biomarkers of radiation-induced brain injury in a rat model of aging. Fischer 344 x Brown Norway rats received fractionated whole-brain irradiation (fWBI rats, 40 Gy, 8 fractions over 4 weeks) or sham-irradiation (Sham-IR rats) at 12 months of age; all analyses were performed at 26-30 months of age. Spatial learning and memory were measured using the Morris water maze (MWM), hippocampal metabolites were measured using proton magnetic resonance spectroscopy ((1)H MRS), and hippocampal glutamate receptor subunits were evaluated using Western blots. Young rats (7-10 months old) were included to control for age effects. The results revealed that both Sham-IR and fWBI rats exhibited age-dependent impairments in MWM performance; fWBI induced additional impairments in the reversal MWM. (1)H MRS revealed age-dependent decreases in neuronal markers, increases in glial markers, but no detectable fWBI-dependent changes. Western blot analysis revealed age-dependent, but not fWBI-dependent, glutamate subunit declines. Although previous studies demonstrated fWBI-induced changes in cognition, glutamate subunits, and brain metabolites in younger rats, age-dependent changes in these parameters appear to mask their detection in old rats, a phenomenon also likely to occur in elderly fWBI patients >70 years of age.

Figure 1

(A) Fischer 344 × Brown Norway rats received fWBI (5Gy twice a week for 4 weeks) or Sham-IR at 12 months, were tested on the Morris water maze (MWM) at 26 months, had their brain metabolites assessed by magnetic resonance spectroscopy (MRS) at 27 months, and had their hippocampal glutamate receptor subunits measured by Western blots (WB) at 30 months. Results were compared with those from 7–10 month old rats. (B) The Morris water maze (MWM) testing schedule for determining the spatial learning, reference memory, and spatial reversal learning ability of old rats that received either fWBI or sham-irradiation at middle age; 7–8 month-old unirradiated rats were tested at the same time as the old rats. For the “standard” version of the MWM, each week consisted of training the rats for 5 days (T-week) to locate the escape platform followed by a single probe trial (P) on the 6th day with the platform lowered beyond their reach. This schedule was repeated 4 times (T-weeks 1–4) over 4 weeks. For the “reversal” version of the MWM, the platform was moved to the opposite quadrant, and the rats trained for 5 days (T) to locate the escape platform followed by a single probe trial on the 6th day with the platform lowered beyond their reach.

Figure 2

Performance of Young, Sham-IR, and fWBI groups on the standard (A–C) and reversal (D–F) versions of the MWM. (A) The total distance to platform during the training trials of the standard MWM version revealed an age effect, but no fWBI effect at old age. The rate of learning was similar in all 3 groups of rats. (B) The mean distance to platform during the probe trials of the standard MWM version again revealed an age effect, but no fWBI effect at old age. (C) Summing the mean distance to platform for probe trials 1–4 of the standard MWM also revealed an age effect, but no fWBI effect at old age. (D) The total distance to platform during the training trials of the reversal MWM revealed an effect of fWBI at old age on Days 4 and 5. (E) The 24-hour saving for the total distance to platform on the reversal MWM also revealed an effect of fWBI. (F) The mean distance to platform on the probe trial of the reversal MWM revealed an age effect, but no fWBI effect at old age.

Figure 3

Western blot analysis of the glutamate receptor subtypes. (A) Representative immunoblots of the NMDA subunits, NR1, NR2A, and NR2B, as well as the AMPA subunits, GluR1 and GluR2, from Young (Y), Sham-IR (S), and fWBI (W) rats; actin was used as the loading control. Quantitation of the individual bands in the gels revealed an age effect, but no fWBI effect at old age for NR1 (B), NR2A (C), NR2B (D), and GluR1 (E). There was no age or fWBI effect for GluR2 (F).

Figure 4

T2 proton magnetic resonance images of a Sham-IR rat brain showing the voxel placement for obtaining the ¹H MR spectra in (A) sagittal, (B) horizontal, and (C) coronal images. The voxels (2.5 mm × 2.5 mm × 2.5 mm) were placed to include the dorsal hippocampus. (D) A representative ¹H MR spectrum from a Sham-IR rat with the peaks for the neuronal markers, glutamate (Glu) and N-acetyl-aspartic acid (NAA) and for the glial markers, myo-inositol (Ins) and glutamine (Gln) identified. Quantitation was performed for the metabolites whose profiles have an SD <20% (Glu, NAA, Ins), or an SD >20%, but <50% (Gln).

Figure 5

Concentrations of the hippocampal metabolites detected by ¹H MRS. The neuronal markers, glutamate (Glu) and N-acetyl-aspartic acid (NAA), revealed a decrease with age, but no effect of fWBI at old age. The glial markers, myo-inositol (Ins) and glutamine (Gln), revealed an increase with age, but no effect of fWBI at old age. *P < 0.05 for comparison of the fWBI and Sham-IR groups.