Figure 1.
Intracerebroventricular injection of AAV2/1-EGFP at different neonatal ages results in distinctive transduction pattern.
Newborn mice were injected with AAV2/1-EGFP into cerebral ventricles at intervals of 6–7 hours till 36 hours postnatal. Representative brain sections from mice aged P21 were stained with anti-EGFP antibody. A representative image of an EGFP expressing neuron (arrowhead) and an astrocyte (arrow) from the cortex is shown (inset, a). Areas of interest, referenced in the manuscript, that show appreciable EGFP expression have been marked as; ctx, cortex; cereb, cerebellum; hpc, hippocampal formation; midbr, midbrain; sn, substantia nigra; str, striatum; olf, olfactory bulb; thal, thalamus; *, lateral ventricle; #, 4th ventricle. (n = 4/group). Scale bar, 500 µm.
Figure 2.
Comparative biodistribution of pseudotyped AAVs injected at different neonatal ages in mice detected by Xenogen IVIS Spectrum fluorescence imager.
Wild type mice were injected into the cerebral ventricles with pseudotyped AAV2/n-EGFP on different neonatal days (P0, P2 and P3) and analyzed on day P21. Representative pseudo color images from each group (standardized to non-injected control brain) show differential fluorescent intensities depending on serotype injected and timing of virus injection. n = 3–4/time point/serotype.
Figure 3.
Immunohistochemical analysis of EGFP biodistribution following intracerebrovcentricular AAV delivery in neonatal mice.
Representative sections of 3 week old mice injected on neonatal day P0, P2 or P3 show that the biodistribution of EGFP is dependent on serotype used and the time of injection. a,g,m, AAV2/1; b,h,n, AAV2/2; c,i,o, AAV2/5; d, j, p, AAV2/7; e, k, q, AAV2/8; f, l, r, AAV2/9; a-f, P0 injection, g-l, P2 injection, m-r, P3 injection.
Table 1.
Comparative summary of the effect of AAV capsid serotype and timing of injection on regional distribution of EGFP expression in neonatal mice.
Figure 4.
AAV2/n-EGFP tropism for neurons following intracerebrovcentricular delivery in neonatal mice.
Representative tricolor merged fluorescent photomicrograph from 3-week-old wild type mice injected on neonatal day P0, P2 or P3 with pseudotyped AAV2/n. Paraffin embedded brain sections were co-labeled with anti EGFP antibody (488 nm-green), anti β-tubulin (568 nm-red) and DAPI counterstain (blue). Images were scanned from the cortex of mice injected at P0 (a-f), P2 (g-l) or P3 (m-r) with AAV2/1 (a,g,m), AAV2/2 (b,h,n), AAV2/5 (c,i,o), AAV2/7 (d,j,p), AAV2/8 (e,k,q) and AAV2/9 (f,l,r). Arrowhead, EGFP expressing neuron; arrow, EGFP expressing astrocyte. n = 3–4/serotype/time of injection. Magnification 400x.
Figure 5.
AAV2/n-EGFP tropism for astrocytes following intracerebroventricular delivery in neonatal mice.
Representative tricolor merged fluorescent photomicrograph from 3 week old wild type mice injected on neonatal day P0, P2 or P3 with pseudotyped AAV2/n. Paraffin embedded brain sections were co-labeled with anti EGFP antibody (488 nm-green), anti GFAP-Cy5 (568 nm-red) and DAPI counterstain (blue). Images were scanned from the cortex of mice injected at P0 (a-f), P2 (g-l), P3 (m-r) with AAV2/1 (a,g,m), AAV2/2 (b,h,n), AAV2/5 (c,i,o), AAV2/7 (d,j,p), AAV2/8 (e,k,q) and AAV2/9 (f,l,r). Arrow, EGFP expressing astrocyte; arrowhead, EGFP expressing neuron. n = 3–4/serotype/time of injection. Magnification 400x.
Table 2.
Comparative summary of the effect of AAV capsid serotype and timing of injection on cellular tropism and intensity of EGFP expression in neonatal mice.