Fig 1.
Summary of Experimental Design.
Green arrows indicate methotrexate (MTX) injection. Blue arrows indicate FDG-PET. Red arrows indicate non-brain directed radiation therapy (NBRT). Sham-treated mice are indicated by CONT. Black arrows indicate sacrifice. Multi-colored arrows indicate multiple procedures occurring within the indicated time frame.
Fig 2.
Non-brain directed radiation results in global brain glucose hypometabolism.
(A) FDG-PET region of interest (ROI) selection subdivided the mouse brain into 19 areas. (B). When [18F]FDG uptake was normalized by blood, significant hypometabolism was noted across all areas, * = p < 0.05. Error bars = standard error of the mean. R/LSTR = Right/Left Striatum; CTX = Cortex; R/LHIP = Right/Left Hippocampus; THA = Thalamus; CB = Cerebellum; BFS = Basal forebrain; HYP = Hypothalamus; R/LAMY = Right/Left Amygdala; BS = Brainstem; CG = Cingulum; SC = Superior colliculi; OLF = Olfactory bulb; R/LMID = Right/Left Midbrain; R/LIC = Right/Left Inferior colliculi.
Fig 3.
The rostral cortex of NBRT-treated mice reveals early astrocytosis and microgliosis.
In the rostral cortex of mice administered NBRT sacrificed at day 3, increased numbers of GFAP+ astrocytes (C, black arrow) were identified as compared to CONT mice (A, black arrow). No significant differences in astrocyte numbers were noted when MTX mice (E, black arrow) were compared to the NBRT mice (C). Increased numbers of Iba1+ microglia (red, white arrow) (D) were identified in NBRT mice as compared to CONT mice (B, gray arrow). No significant differences were noted when MTX mice (F, white arrow) were compared to the NBRT mice (D, white arrows). In contrast to the small, minimally ramified microglia in the CONT mice (B, gray arrows), the microglia in the NBRT and MTX treated mice demonstrated features consistent with activation (D and F, white arrows).
Fig 4.
The striatum of NBRT treated mice reveals early astrocytosis and microgliosis.
In the striatum of the mice administered NBRT sacrificed at day 3, increased numbers of GFAP+ astrocytes (C, black arrow) were identified as compared to CONT mice (A, black arrow). No significant differences in astrocytes were noted when MTX mice (E, black arrow) were compared to the NBRT mice. Increased numbers of Iba1+ microglia (D, white arrows) were identified in the NBRT mice as compared to CONT mice (B, gray arrows). No significant differences were noted when MTX mice (F, white arrows) were compared to the NBRT mice (D). In contrast to the small, minimally ramified microglia in the CONT mice (B, gray arrows), the microglia in the NBRT and MTX treated mice demonstrated features consistent with activation (D and F, white arrows).
Fig 5.
The cerebellum of NBRT treated mice reveals early astrocytosis and microgliosis.
In the cerebellum of the mice administered NBRT sacrificed at day 3, increased numbers of GFAP+ astrocytes (C, black arrow) were identified as compared to CONT mice (A, black arrow). No significant differences in astrocytes were noted when MTX mice (E, black arrow) were compared to NBRT mice. Increased numbers of Iba1+ microglia (D, white arrows) were identified as compared to CONT mice (B, gray arrows). No significant differences were noted when MTX mice (F, white arrows) were compared to the NBRT mice (D). In contrast to the small, minimally ramified microglia in the CONT mice (B, gray arrows), the microglia in the NBRT and MTX treated mice demonstrated features consistent with activation (D and F, white arrows).
Fig 6.
Non-brain directed radiation therapy results in a multifocal, early and persistent astrocytosis and microgliosis.
(A) Log (Average) Number of GFAP+ cells in NBRT (sacrifice days 3 and 30), MTX, and CONT (C) mice. * = p < 0.05, ** = p < 0.01, compared to controls; § = p < 0.05, compared to NBRT Day 30. (B) Log (Average) Number of Iba1+ cells in NBT (sacrifice days 3 and 30), MTX, and CONT (C) mice. * = p < 0.05, ** = p < 0.01, compared to controls; § = p < 0.05, compared to NBRT Day 30.
Fig 7.
The hippocampus of NBRT treated mice demonstrates early microgliosis, but not astrocytosis.
In the hippocampus of the mice administered NBRT sacrificed at day 3, there was no statistical difference in the numbers of GFAP+ astrocytes (C, black arrow) as compared to CONT mice (A, black arrow). No significant differences were noted when MTX mice (E, black arrow) were compared to the NBRT mice. Increased numbers of Iba1+ microglia were identified in the NBRT mice (D, white arrows) as compared to CONT mice (B, gray arrows). No significant difference in astrocyte numbers were noted when MTX mice (F, white arrows) were compared to NBRT mice (D). In contrast to the small, minimally ramified microglia in the CONT mice (B, gray arrows), the microglia in the NBRT and MTX treated mice demonstrated features consistent with activation (D and F, white arrows).
Fig 8.
The medulla of NBRT treated mice demonstrates early microgliosis, but not astrocytosis.
In the medulla of the mice administered NBRT sacrificed at day 3, there was no statistical difference in the numbers of GFAP+ astrocytes (C, black arrow) as compared to CONT mice (A, black arrow). No significant difference in astrocyte numbers were noted when MTX mice (E, black arrow) were compared to the NBRT mice. Increased numbers of Iba1+ microglia were identified in the NBRT mice (D, white arrows) as compared to CONT mice (B, gray arrows). No significant differences in microglia numbers were noted when MTX mice (F, white arrows) were compared to NBRT mice (D). No appreciable difference in microglia morphology was noted between the CONT (B, gray arrows), NBRT, and MTX mice (D and F, white arrows).
Fig 9.
The caudal cortex of NBRT treated mice demonstrates early astrocytosis, but not microgliosis.
In the caudal cortex of the mice administered NBRT sacrificed at day 3, increased numbers of GFAP+ astrocytes (C, black arrow) were identified as compared to CONT mice (A, black arrow). No significant differences in astrocyte numbers were noted when MTX mice (E) were compared to NBRT mice. Similarly not significant differences in Iba1+ microglia (D, white arrows) numbers were identified as compared to CONT mice (B, gray arrow). No significant differences in microglia numbers were noted when MTX mice (F) were compared to the NBRT mice (D). In contrast to the small, minimally ramified microglia in the CONT mice (B, gray arrows), the microglia in the NBRT and MTX treated mice demonstrated features consistent with activation (D and F, white arrows).
Fig 10.
The NBRT treated mice demonstrate persistent multifocal astrocytosis.
In NBRT mice sacrificed at day 30, increased numbers of GFAP+ astrocytes were identified in the rostral cortex, caudal cortex, and striatum (A-C, black arrows).
Fig 11.
The NBRT treated mice demonstrate persistent multifocal microgliosis.
In NBRT mice sacrificed at day 30, increased numbers of Iba1+ microglia were identified in the cerebellum and medulla (A-B, white arrows).
Fig 12.
NBRT and MTX mice demonstrate increased hippocampal TNF-α expression.
Increased TNF-α immunoreactivity (red) was identified in the hippocampus of NBRT (B, black arrows) and MTX (D, black arrows) mice sacrificed at day 3 as compared to CONT (A, black arrow). In contrast, the level of TNF-α immunoreactivity in mice administered NBRT sacrificed at day 30 (C, black arrow) appears similar to CONT (A).