Research ArticleNEUROSCIENCE

Physical positioning markedly enhances brain transduction after intrathecal AAV9 infusion

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Science Advances  14 Nov 2018:
Vol. 4, no. 11, eaau9859
DOI: 10.1126/sciadv.aau9859
  • Fig. 1 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).

  • Fig. 2 Inversion and rotation improve the extent and consistency of gene transfer to cerebral cortex and basal forebrain after intrathecal AAV9 infusion.

    (A) GFP labeling in medial entorhinal cortex of every animal in the study after intrathecal infusion. GFP expression among rats that remained upright after intrathecal AAV9 infusion was highly variable and minimal in some animals. Gene expression in rats that were inverted or rotated for 2 hours after surgery was both more extensive and more consistent, with robust entorhinal transduction in all 14 animals. Rostral is left in all images. Scale bar, 500 μm. (B to I) Inversion and rotation significantly and substantially enhanced gene transfer to all regions of cortex and basal forebrain. n = 7 rats per cohort. ****P ≤ 0.0001, ***P ≤ 0.001, **P ≤ 0.01: significance of comparison to upright group by one-way analysis of variance (ANOVA) and Tukey’s post test. No significant differences were detected between inverted and rotated rats. Error bars represent SD. Y axes are plotted on a logarithmic scale.

  • Fig. 3 AAV9-mediated gene transfer in the brain is predominately targeted to neurons.

    (A to D) Intrathecal AAV9 infusion and inversion for 2 hours: (A) 95.5% of GFP-labeled cells are neurons, by colocalization with neuronal nuclei (NeuN); (B) 0.779% of GFP-labeled cells are astrocytes, by colocalization with glial fibrillary acidic protein (GFAP) and sex-determining region Y-box9 (Sox9); and (C) 4.39% of GFP-labeled cells are oligodendrocytes, by colocalization with adenomatous polyposis coli (APC). (D) No microglia were transduced by colocalization with ionized calcium-binding adapter molecule 1 (Iba1). Scale bars, 50 μm.

  • Fig. 4 Inversion and rotation enhance gene transfer to cervical DRG.

    In rats that were either inverted or rotated after intrathecal AAV9 infusion, significantly more cervical DRG cells were transduced than in rats that remained upright. Nearly all transduced cells were neurons by colocalization with CaMKII (99.5%), including both small- and large-diameter DRG neurons. Scale bar, 500 μm.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/11/eaau9859/DC1

    Fig. S1. Intrathecal AAV9 infusion does not cause microglial activation or astrogliosis.

    Fig. S2. Inversion and rotation increase gene transfer to cervical spinal cord but not to other spinal levels.

    Fig. S3. Transduced cells are evenly distributed after intracortical AAV9 injection.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Intrathecal AAV9 infusion does not cause microglial activation or astrogliosis.
    • Fig. S2. Inversion and rotation increase gene transfer to cervical spinal cord but not to other spinal levels.
    • Fig. S3. Transduced cells are evenly distributed after intracortical AAV9 injection.

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