3. Results
3.7 Absense of brain pathology in Stop-Nrg1*NEX-Cre and Stop-Nrg1*CKII-Cre mice
Overexpression of growth- and differentiation factors could lead to abnormal development, neuroinflammation or cell death. Stop-Nrg1*NEX-Cre and Stop-Nrg1*CKII-Cre mice displayed normal cage behavior compared to control littermates.
To exclude neuroinflammation or other neuropathology in these mice, we performed chromogenic immunostainings for markers of neuropathology and inflammation on coronal paraffin sections at 4 months and one year of age.
Hematoxylin-Eosin (H+E) staining revealed no gross abnormalities in brain morphology in both transgenic models (data not shown). Subsequently we tested for astrogliosis or microgliosis by chromogenic immunostainings for GFAP, IBA1 and MAC3 on coronal paraffin sections (bregma -1.7) (Eng and Ghirnikar, 1994; Hanisch and Kettenmann, 2007). Furthermore, we examined brain sections for axonal swellings by staining for amyloid precursor protein (APP), and T-cell infiltration by staining for the T-cell antigen CD3.
Stainings for activated microglia, T-cell infiltration, and axonal swellings showed no signs of pathology in Stop-Nrg1*NEX-CreandStop-Nrg1*CKII-Cre mice at 4 months and 1 year of age compared to controls (Fig. 17). Immunostainings for the astrocytic marker GFAP were quantified with a semi-automated method, which determined the GFAP+ area in relation to the region of interest. Hippocampus and fimbria were analyzed as examples for grey and white matter regions, respectively (Fig. 18A). No obvious differences in GFAP+ area were observed in the hippocampus of Stop-Nrg1*NEX-Cre or Stop-Nrg1*CKII-Cre mice. However, the GFAP+ area in the fimbria at 4 months was significantly larger in Stop-Nrg1*NEX-Cre (13.41 % 1.49) than in Stop-Nrg1*CKII-Cre mice (5.76 % 1.84; *p < 0.05). This effect however, was not significant when compared to control mice (Fig. 18B). At 1 year of age there was no significant difference detectable in any of the mice tested (Fig. 18C), indicating a non-progressive pathology. Taken together, Stop-Nrg1*NEX-Cre and Stop-Nrg1*CKII-Cre mice showed no signs of progressive astrogliosis (Fig. 18B, C).
Fig. 17: Stop-Nrg1*NEX-Cre and Stop-Nrg1*CKII-Cre mice show no signs of neuroinflammation. (A) Chromogenic immunostainings for neuropathology markers on coronal paraffin sections (bregma -1.7) of 4 months old Stop-Nrg1*NEX-Cre, Stop-Nrg1*CKII-Cre and control mice for activated microglia (MAC-3), T-cell infiltration (CD3) and axonal swellings (APP), reveal no signs of neuropathology or -inflammation. Insets in CD3 stainings show high magnifications of occasionally found CD3+ T-cells (indicated by boxes in overviews). Sections of CNP-/- mice, which received cryo lesions, were used as positive controls (Wieser et al., 2013). Scale bars, 50 µm (overviews), 100 µm (CD3), 10 µm (insets).
(B) Sustained HA-NRG1 overexpression does not lead to neuroinflammation in aged Stop-Nrg1*NEX-Cre and Stop-Nrg1*CKII-Cre mice. Chromogenic immunostainings on coronal paraffin sections from 1 year old mice for MAC-3, CD3 and APP, as in (A). CA3, hippocampal CA3 region; CC, corpus callosum; Fim, fimbria; SO, stratum oriens; SP, stratum pyramidale;
SR, stratum radiatum. Scale bars, 100 µm (overviews), 20 µm (insets).
Likewise, no changes in activated microglia were found in immunostainings for MAC-3 (Fig. 17). To determine microglia numbers, immunostaining for IBA1 was
performed (Fig. 19). Similar to GFAP immunostainings, a semi-automated quantification of chromogenic IBA1 immunostainings was conducted. Cortex and hippocampus were quantified as examples for grey matter, and the corpus callosum as an example for white matter regions (quantified regions are illustrated in Fig. 19A, C). At 4 months of age no difference in IBA1+ area was detectable in both transgenic models. A trend for increased IBA1+ area in the cortex of NEX-Cre controls and Stop-Nrg1*NEX-Cre mice was not significant (Fig. 19B). Also at 1 year of age no significant differences in IBA1+ area were detected in cortex and hippocampus of Stop-Nrg1*NEX-Cre and Stop-Nrg1*CKII-Cre mice. However, there was an increase in IBA1+ area in the hippocampus of NEX-Cre control mice (7.49 % 1.34) compared to CKII-Cre and Cre mice (CKII-Cre: 3.03 % 0.56; Stop-Nrg1*CKII-Cre: 3.23 % 0.35; *p < 0.05), and Stop-Nrg1 mice (2.95 % 0.29; *p < 0.01), but not to Stop-Nrg1*NEX-Cremice (Mean: 4.46 % 1.62), indicating an effect of NEX haploinsufficiency (Fig. 19D).
Taken together, chronically elevated HA-NRG1 expression has no potent neurodegenerative or -inflammatory effects when overexpression starts at postnatal or even embryonic stages. However, NEX (NeuroD6) haploinsufficiency in heterozygous NEX-Cre driver mice is associated with mildly increased microgliosis in the hippocampus of aged mice. HA-NRG1 overexpression in Stop-Nrg1*NEX-Cre mice seemed to be beneficial and counteracted this effect.
Fig. 18: Stop-Nrg1*NEX-Cre and Stop-Nrg1*CKII-Cre mice show no signs of astrogliosis. (A) Chromogenic immunostaining for astrocytes (GFAP) on coronal paraffin sections from 4 months old Stop-Nrg1*NEX-Cre, Stop-Nrg1*CKII-Cre and control mice (bregma -1.7). Dashed lines mark quantified areas. Square indicates position of high magnification image. Fim, fimbria; Hipp, hippocampus. Scale bars, 500 µm. (B) Semi-automated quantification of GFAP+ area in hippocampus and fimbria of Stop-Nrg1*NEX-Cre, Stop-Nrg1*CKII-Cre and control mice at 4 months of age reveals a significant increase in GFAP+ area in the fimbria of Stop-Nrg1*NEX-Cre mice compared to Stop-Nrg1*CKII-Cre mice, that, however, does not differ significantly from control mice. (*p < 0.05, one-way ANOVA; Bonferroni’s multiple comparison test; n.s., not significant; n-numbers indicated in the graphs). (C) Semi-automated quantification of GFAP+ area in hippocampus and fimbria of Stop-Nrg1*NEX-Cre, Stop-Nrg1*CKII-Cre and control mice at 1 year of age confirms absence of astrogliosis. (One-way ANOVA with Bonferroni’s multiple comparison test; n.s., not significant; n-numbers indicated in the graphs).
Fig. 19: Stop-Nrg1*NEX-Cre and Stop-Nrg1*CKII-Cre mice show no signs of microgliosis. (A) Chromogenic immunostaining for microglia (IBA1) on coronal paraffin sections from 4 months old Stop-Nrg1*NEX-Cre, Stop-Nrg1*CKII-Cre and control mice (bregma -1.7). Dashed lines mark quantified areas. Dashed square in thresholded black/white image indicates position of high magnification images (Asterisk). Cc, corpus callosum; Cx, cortex; Hipp, hippocampus. Scale bars, 500 µm (overvies), 100 µm (high magnifications). (B) Semi-automated quantification of IBA1+ area in cortex, hippocampus and corpus callosum of Stop-Nrg1*NEX-Cre, Stop-Nrg1*CKII-Cre and control mice at 4 months of age reveals absence of microgliosis. (One-way ANOVA; Bonferroni’s multiple comparison test; n.s., not significant. n-numbers indicated in the graphs). (C) Chromogenic immunostaining for IBA1 on 1 year old animals as in (A). Scale bars, 500 µm (overviews), 100 µm (high magnifications).
(D) Semi-automated quantification of IBA1+ area in 1 year old animals as in (B), reveals significantly increased IBA1+ area in the hippocampus of NEX-Cremice. Note the moderate increase of IBA1+ area in Stop-Nrg1*NEX-Cre mice, indicating an effect of NEX haploinsufficiency. (One-way ANOVA with Bonferroni’s multiple comparison test; n.s., not significant; n-numbers indicated in the graphs).