Table 1.
Antibodies used in immunohistochemistry.
Figure 1.
Integrity of purified AAV6-EGFP and AAV8-EGFP vectors.
Purified AAV6-EGFP (A) and AAV8-EGFP (B) vectors were negatively stained with uranyl acetate and examined by transmission electron microscopy. Empty particles (arrows) can be distinguished on the basis of the electron dense center and are indicated by arrows. Properly DNA-filled particles made up 95% of the total particles in each case. Silver stain (C) was performed on SDS-PAGE after loading equally with 5×109 genome-containing particles of purified AAV6-EGFP and AAV8-EGFP. This revealed 3 virion protein bands of Vp1, Vp2, and Vp3, with molecular weight of 87, 72 and 62 KDa, respectively, in each sample, corresponding to the particular AAV capsid proteins revealed by immunoblot using AAV specific antibody (D). The absence of other protein bands demonstrates the high purity of the preparations.
Figure 2.
AAV6-EGFP and AAV8-EGFP transduction in DRG.
DRG sections 3 weeks after AAV6-EGFP injection (A) or AAV8-EGFP injection (B) were immunostained with antibodies to GFP and the indicated markers. Co-localization is shown in the merged images. Arrowheads point to the same neuron when stained for EGFP, the specified marker, and in the merged image. Scale bars: 100 µm for all images. DRG neuronal size histograms 3-weeks following DRG injection of AAV6-EGFP (C) and AAV8-EGFP (D) show the distribution frequencies for transduced somata (green bars) compared to the total DRG neuron population identified by β3-tubulin staining (red bars).
Figure 3.
Bar graphs represent the percentage of in vivo transduction rates per total DRG neurons of AAV6-EGFP and AAV8-EGFP 3 weeks (n = 8 DRGs from 4 rats per vector) and 3 months (n = 8 DRGs from 4 rats per vector) after injection. Two-way ANOVA identified a main effect of vector (p<0.001) and significant paired comparisons at each time point (*, p<0.05). Data are presented as mean ± SD.
Figure 4.
Central termination patterns of EGFP-positive axons in the spinal cord.
Representative low-power views of transverse spinal cord sections at fourth and fifth lumbar levels show EGFP (green) expression 3 weeks following injection of AAV6-EGFP (A) and AAV8-EGFP (B). Yellow lines indicate the spinal cord transverse section profiles and white dashed lines indicate cord grey matter. Additional images show co-labeling of EGFP (green), CGRP (red), CaMKII (red), and NeuN (red) in the dorsal horn for AAV6-EGFP (A1, A2, and A3) and AAV8-EGFP (B1, B2 and B3). White dashed lines indicate dorsal horns, with laminae I-III marked in each section. The boxed areas in A3 and B3 are shown at higher magnification, which demonstrate that vectors AAV6-EGFP (A4) and AAV8-EGFP (B4) transduced no dorsal horn neuronal somata immunolabeled by NeuN. Scale bars: A and B, 500 µm; A1, A2, A3, B1, B2, and B3, 100 µm; A4 and B4, 50 µm.
Figure 5.
EGFP expression in sciatic nerve and cutaneous tissue.
Longitudinal sciatic nerve sections (A) reveal immunofluorescence for EGFP expression (green) in a subpopulation of β3-tubulin-positive fibers (red) 3 weeks after intraganglionic injection of AAV6-EGFP (top) and AAV8-EGFP (bottom). In the medial plantar skin (B, low power on left, higher power on right), AAV6-EGFP sections (top) show numerous thick bundles of EGFP-positive nerve fibers (green; Hoechst counterstain blue) in the subcutaneous layer (arrows), and EGFP-positive thinner nerve fibers were also observed in the dermis and dermoepidermal junction (arrowheads). However, AAV8-EGFP sections (bottom) show only few EGFP-positive fibers observed in the subcutaneous layer (arrow) and dermis (arrowhead). Labels: E, epidermis; D, dermis; and SC, subcutaneous layer.
Figure 6.
Identification of inflammatory response and neurotoxicity in AAV injected DRGs.
A. Representative examples of GFAP (top row), GS (second row), Iba1 (third row), and ATF3 (bottom row) immunoreactivity in dorsal root ganglia 4 weeks after injection of AAV6-EGFP (first column), AAV8-EGFP (second column), and control condition (third column), which is saline injection for GFAP, GS, and Iba1, but spinal nerve ligation (SNL) as a positive control for ATF3. Samples are co-stained for EGFP immunoreactivity to identify transduced neurons. AAV-injected DRG neurons exhibit significantly less ATF3-positive nuclei in comparison to the SNL group. Scale bar: 50 µm. B. Quantification of immunoreactivity for each marker. For GFAP and GS, the measurement is the ratio of satellite glial cell area (identified by GFAP or GS) divided by the area of the neuron they surround. For Iba1, the area of immunopositive microglia (identified by Iba1) as a fraction of the total field is recorded. For ATF3, the percent of positive cells is recorded. The number in each column is the number neurons for each group for GFAP, GS, and ATF3, and fields for Iba1, which were derived from 4–7 different sections and at least 4 animals per group. Data are presented as mean ± SD. *p<0.05, ***p<0.001.
Figure 7.
Identification of inflammatory response in dorsal horn (DH) of AAV injected rats.
Representative sections (A) revealed no differences in astrocyte activation (GFAP and GS), or activation of resident microglia or infiltration of hematogenous macrophages (Iba1) between cord tissues from animals in which dorsal root ganglia were injected with AAV6-EGFP or AAV8-EGFP compared to saline injected control rats. Scale bar: 100 µm. Quantification of GFAP, GS, and Iba1 immunofluorescence (B) was done along lines overlaid from central canal to dorsal root entry zone (DREZ), producing traces of intensity. The averaged fluorescence intensities across the length of each trace (C) were similar for all groups. The number in each bar is the number of analyzed sections per group from at least 4 animals per group. Data are presented as mean ± SD. There were no differences between groups.
Figure 8.
Pain behavior evaluation after intraganglionic AAVs.
The response to innocuous punctate mechanical stimulation (von Frey, A) and hyperalgesia behavior after touch with a pin (Pin, B) following vehicle or vector injections evaluated across 4 weeks following vector injection. Left panels show the time course of testing performed upon the plantar surface of the right paw ipsilateral to injection of either vehicle (square), AAV6-EGFP (triangle) or AAV8-EGFP (diamond) into the fourth lumbar (L4) and L5 DRGs. Results are means ± SD (n = 6 animals in each group). For ANOVA main effects, *p<0.05, **p<0.01, and ***p<0.001. For differences between the test day and baseline at day 0 (d0), ††p<0.01, †††p<0.001. In the right panels, the time course data are analyzed using area under the curve (AUC) across the 4-week testing period for measures normalized to d0, allowing comparisons between vectors. Results are means ± SD. There were no differences between groups.