Journal: Science Advances
Article Title: Physical positioning markedly enhances brain transduction after intrathecal AAV9 infusion
Figure Lengend Snippet: Inversion and rotation greatly enhance gene transfer to the brain after intrathecal AAV9 infusion. ( A to C ) GFP labeling in 40-μm-thick sagittal brain sections in rats that received intrathecal AAV9-CAG-eGFP, and (A) remained upright for 2 hours, (B) were inverted for 2 hours or (C) were rotated for 2 hours after surgery. From left to right, sections are 0.5, 2.5, and 4.5 mm lateral from bregma. Both inversion and rotation substantially increased gene transfer to entorhinal (e), prefrontal (pf), frontal (f), parietal (pa), and limbic (li) cortices, as well as hippocampus (hp), basal forebrain (bf), cerebellum (cb), and olfactory bulb (ob). There was minimal gene transfer to striatum (st), thalamus (th), hypothalamus (hy), and brainstem (bs). Scale bar, 2 mm. ( D ) The average increase in the number of GFP-positive cells per section in inverted or rotated animals relative to upright animals is shown for nine brain regions: prefrontal, frontal, parietal, entorhinal, and limbic cortices, as well as hippocampus, subiculum, horizontal diagonal band, and medial septum/vertical diagonal band. Inversion and rotation increased the number of GFP-positive cells by an average of 1520 and 1890%, respectively, relative to upright animals. This increase was highly significant as determined by Friedman test ( P = 0.0003) and Tukey’s post tests. Error bars represent the 95% confidence interval. ** P ≤ 0.01. ( E ) Following inversion for 2 hours, 73.0% of transduced neurons in the entorhinal cortex are excitatory glutamatergic neurons, and 27.0% are inhibitory GABAergic neurons (arrow). The image is a single optical section acquired with structured illumination. ( F ) In the cholinergic basal forebrain, inversion for 2 hours induces GFP expression in 22.5% of cholinergic neurons in the horizontal diagonal band and ( G ) 13.9% of cholinergic neurons in the medial septum/vertical diagonal band (MS/VDB), based on colocalization with p75. Scale bars, 100 μm (E to G).
Article Snippet: The following antibodies were used for fluorescent staining of brain: chicken polyclonal anti-GFP (1:1000; Aves Labs #GFP-1020), rabbit polyclonal anti-GFP (1:1000; Invitrogen Thermo Fisher Scientific #A6455), goat polyclonal anti-Sox9 (1:500; R & D Systems #AF3075), chicken polyclonal anti-GFAP (1:2000; EnCor Biotechnology #CPCA-GFAP), mouse monoclonal anti-APC (1:400; MilliporeSigma #OP80), mouse monoclonal anti–myelin basic protein (MBP; 1:1000; EnCor Biotechnology #MCA-7G7), rabbit polyclonal anti-Iba1 (1:800; Wako #019-19741), rabbit polyclonal anti-NeuN (1:500; Biosensis #R3770), guinea pig polyclonal anti-VGluT1 (1:1000; MilliporeSigma #AB5905), guinea pig polyclonal anti-VGluT2 (1:1000; MilliporeSigma #AB2251), rabbit polyclonal anti-GABA (1:500; MilliporeSigma #A2052), mouse monoclonal anti–rat endothelial cell antigen-1 (RECA-1) (1:100; Abcam #ab9774), mouse monoclonal anti–tyrosine hydroxylase (TH) (1:1000; MilliporeSigma #MAB318), rabbit polyclonal anti-serotonin [5-hydroxytryptamine (5-HT)] (1:500; ImmunoStar #20080), and mouse monoclonal anti-p75 (1:1000; MilliporeSigma #MAB365).
Techniques: Labeling, Expressing, Mass Spectrometry