Fig. 2: AQP4-mRuby3 expression in heterozygous mice does not affect neuroinflammation.
From: In vivo imaging of the barrier properties of the glia limitans during health and neuroinflammation
a Scheme of aEAE experiment. MOG = myelin oligodendrocyte glycoprotein; PTx = pertussis toxin. b Overall aEAE disease severity from day of immunization (day 0) to day 29 determined by the area under the curve (AUC) of female WT and heterozygous (Het) Aqp4-mRuby3 reporter mice (3 independent experiments, n(WT) = 27 and n(Het)=28). SuperPlots83 are shown, where each point represents an individual mouse and outlined points represent the mean of each experiment. Data shown as mean ± SD and analyzed using a two-tailed paired t-test. c, d Water content (in %) after drying process of 24 h in brains (c) and spinal cords (d) of female heterozygous Aqp4-mRuby3 and WT control mice at different clinical stages of aEAE, from 3 independent experiments. n(WT Naïve)=8, n(Het Naïve)=7; n(WT onset)=6, n(Het onset)=6; n(WT peak)=5, n(Het peak)=5; n(WT chronic)=4, n(Het chronic)=3. Data shown as mean ± SD and analyzed using two-way ANOVA with Šidák’s multiple comparisons test. e Splenocytes from female WT (n = 5) and Het Aqp4-mRuby3 mice (n = 3) were isolated at day 30 of aEAE. Incorporation of 3H-thymidine as a measure for cell proliferation in counts per minute (CPM) of splenocytes in medium (RM) as a negative control, in the presence of 10, 20, and 40µg/mL of MOG35-55, or polyclonal anti-CD3 and CD28 antibody induced polyclonal proliferation as a positive control. Data shown as mean ± SD and analyzed by unpaired parametric t-tests. Source data are provided as a Source Data file. f, g Immunofluorescence staining of 20 µm thick cryosections from female Aqp4-mRuby3 mice during aEAE (n = 2). f Staining for AQP4 in mouse skull cryosection at peak of aEAE (score 1). Pink arrowhead shows the loss of the perivascular polarized AQP4-mRuby signal during neuroinflammation. g, h Laminin (magenta) and CD45 (green) immunofluorescence stainings of brain cryosection of mouse at chronic stage of aEAE (score 0.5 to 1). h Quantification of perivascular cuffs in WT (n = 3) and Aqp4-mRuby3 mice (n = 2) from 1 aEAE experiment. Top panel shows representative images of small and large perivascular cuffs observed in immunofluorescence stainings from (g). Data shown as mean ± SD, two-way ANOVA with Šídák multiple comparisons test was performed.
