96 Chapter 4 Figure 5. TSPO is increased in microglia in SOD1G93A mice but not in ALS spinal cord. a-c Representative images of TSPO expression in microglia and astrocytes in ALS spinal cord. d-f An increase was observed in microglia (P < 0.0001, t=7.445, df=19), HLA-DR+ microglia (P < 0.0001, t=6.007, df=19), and astrocytes (P < 0.0001, t=9.024, df=19) in ALS spinal cord when compared to controls. g A 2.5-fold increase of TSPO+ cells (P < 0.0001, t=12.88, df=19) was observed in the ALS spinal cord. h,i Up to a 3.4-fold increase in the density of TSPO+ microglia (TSPO+IBA1+ cell, P < 0.0001, t=7.541, df=19) (TSPO+HLA-DR+ cells, P < 0.0001, t=3.368, df=19) was observed. j TSPO+ astrocytes were significantly increased (P < 0.0001, t=11.77, df=19) in the spinal cord of ALS patients. k The increase in activated microglia and astrocytes was not associated with an increase in TSPO expression in microglia (P = 0.7684, t=0.3046, df=8) or in astrocytes (P = 0.5047, t=0.6985, df=8). l,m Representative images of TSPO expression in microglia and astrocytes in SOD1G93A spinal cord. n An increase was observed in microglia in SOD1G93A spinal cord when compared to controls at 16 weeks (P=0.0115, t=3.395, df=7) but not at 10 weeks (P = 0.5334, t=0.6509, df=8). o An increase for astrocytes was observed for both 10 weeks (P = 0.0024, t=4.362, df=8) and 16 weeks (P = 0.0248, t=2.848, df=7) p An increase in TSPO+ cells was observed at 10 weeks (P = 0.0011, t=4.931, df=8) but not 16 weeks (P = 0.7299, t=0.3594, df=7). q No increase in the number of TSPO+ microglia was observed (10 weeks: P = 0.5244, t=0.6656, df=8; 16 weeks, P = 0.0930, t=1.944, df=7). r TSPO+ astrocytes were increased up to 15-fold in the spinal cord of SOD1G93A mice (10 weeks: P = 0.0003, t=6.085, df=8; 16 weeks: P = 0.382, t=2.548, df=7). s Despite no increase in the number of TSPO+ microglia, an increase in the amount of TSPO per cell was observed in microglia (P = 0.0451, t=2.435, df=7), but not astrocytes (P = 0.4052, t=0.8856, df=7). Statistical significance in d-k, and o-s was determined by a two-tailed unpaired t-test. Box and whiskers mark the 25th to 75th percentiles and min to max values, respectively, with the median indicated. Scale bar = 50µm, inserts are digitally zoomed in (200%). SOD1G93A miceexpress high levels ofmutant SOD1 that initiates adult-onset neurodegeneration of spinal cord motor neurons leading to paralysis, and as such these mice have been used as a preclinical model for ALS20. To determine the extent to which TSPO+ cells were present in SOD1G93A mice TSPO+ microglia and astrocytes were quantified with immunohistochemistry in the white and grey matter of the spinal cord (Fig. 5l,m). An increase was observed in the total number of microglia (Fig. 5n) and astrocytes (Fig. 5o) in 16-week old SOD1G93A mice but not in 10 week old animals (Fig. 6c,d). The density of TSPO+ cells was increased 2- to 3-fold in presymptomatic disease (10 weeks) compared to non-transgenic littermate control mice in both white and grey matter (Fig. 5p). Increases in the density of TSPO+IBA+ cells were not observed in SOD1G93A mice compared to control animals (Fig. 5q). However, a significant 8- to 15-fold increase in the density of TSPO+GFAP+ astrocytes was observed in 10- and 16week old SOD1G93A mice compared to 10- and 16-week old wild-type mice (Fig. 5r). Finally, we then quantified TSPO+ area in microglia and astrocytes as an index of individual cellular TSPO expression. In contrast to the human data, where there was no change in disease samples relative to controls, expression of TSPO in individual cells was increased by 1.5-fold in microglia in the rodent model. As with the AppNL-G-F and TAUP301S mice above, TSPO expression within astrocytes was unchanged (Fig. 5s). In summary, consistent with the data from AD and relevant mouse models, we have shown that TSPO expression is increased within microglia from SOD1G93A mice, but not increased in microglia from human ALS tissue. TSPO also was unchanged in astrocytes from the SOD1G93A mice and the human disease relatively to those in the healthy control tissues. Increased myeloid cell TSPO expression is found in mouse EAE, but not in MS or marmoset EAE Having found no evidence of increased TSPO expression in activated microglia in human neurodegenerative diseases affecting the brain or spinal cord, we next examined MS as an example of a classical neuroinflammatory disease characterised by microglia with a highly activated pro-inflammatory phenotype. We compared data from human postmortem MS brain (Table S3) to mice with EAE (Table S4). We also examined brain tissue from marmoset
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