Transient demyelination increases the efficiency of retrograde AAV transduction

Abstract

Adeno-associated virus (AAV) is capable of mediating retrograde viral transduction of central and peripheral neurons. This occurs at a relatively low efficiency, which we previously found to be dependent upon capsid serotype. We sought to augment retrograde transduction by providing increased axonal access to peripherally delivered AAV. Others have described utilizing full transection of peripheral nerves to mediate retrograde viral transduction of motor neurons. Here, we examined the ability of a transient demyelinating event to modulate levels of retrograde AAV transduction. Transient demyelination does not cause lasting functional deficits. Ethidium bromide (EtBr)-induced transient demyelination of the sciatic nerve resulted in significant elevation of retrograde transduction of both motor and sensory neurons. Retrograde transduction levels of motor neurons and heavily myelinated, large-diameter sensory neurons increased at least sixfold following peripheral delivery of self-complementary AAV serotype 1 (scAAV1) and serotype 2 (scAAV2), when preceded by demyelination. These findings identify a means of significantly enhancing retrograde vector transport for use in experimental paradigms requiring either retrograde neuronal identification and gene expression, or translational treatment paradigms.

Figure 1

EtBr injection increases the retrograde transduction efficiency of AAV. (a) GFP immunoreactive retrogradely transduced motor neurons in the lumber enlargement following sciatic injection of scAAV1. (b) Higher magnification stacked images from a. (c) Gold (III) chloride staining of transverse sciatic nerve sections demonstrates loss of myelin staining 3 weeks after 0.1% EtBr injection (black indicates myelin staining). (d) Increases in retrograde transduction following demyelination are indicated for each animal (in gray) with the mean values indicated by dashed lines ± SEM (brackets). Lines connect values from the same animals, comparing retrograde transduction efficiency of motor neurons after vector injection in the nondemyelinated sciatic nerve (PBS-injected) to transduction efficiency after vector injection in the demyelinated sciatic nerve (EtBr-injected). Both serotype 1 and 2 exhibit similar increases in retrograde transduction (scAAV1 n = 5, scAAV2 n = 3). Bar = 500 µm (a), 25 µm (b,c). ETBr, ethidium bromide; PBS, phosphate-buffered saline; scAAV1, self-complementary AAV serotype 1; scAAV2, self-complementary AAV serotype 2.

Figure 2

Temporal relationship of EtBr injection to retrograde transduction efficiency. (a) Quantification of pixel density of gold (III) chloride myelin staining in transverse sciatic nerve sections demonstrates a reduction in myelination over time following EtBr injection (ANOVA, P < 0.0001). (b) As demyelination progresses, there is a corresponding increase in the number of retrogradely transduced motor neurons in the lumbar enlargement with scAAV1-eGFP delivery to the sciatic nerve (ANOVA, P < 0.0001). ANOVA, analysis of variance; eGFP, enhanced green fluorescent protein; EtBr, ethidium bromide; scAAV1, self-complementary AAV serotype 1.

Figure 3

EtBr injection increases retrograde transduction of sensory neurons. (a) Incoming sensory afferents projecting to the dorsal horn of the lumbar enlargement demonstrate robust GFP immunoreactivity following scAAV1-GFP retrograde transduction of 0.1% EtBr-injected sciatic nerve. (b,c) Retrogradely transduced sensory neurons in L5 dorsal root ganglia following scAAV1-GFP injection, 1 week after (b) PBS control injection or (c) 0.1% EtBr injection into the sciatic nerve. (d) Quantification of retrogradely transduced sensory neurons within L4/5 dorsal root ganglia illustrates significant increases in retrograde transduction of sensory neurons following EtBr injection (paired t-test; *P < 0.05, n = 4). (e) The proportion of retrogradely transduced sensory neurons immunoreactive for NF200 increases following EtBr injection (paired t-test; *P < 0.05, n = 4). Bar = 500 µm (a), 100 µm (b,c). DRG, dorsal root ganglion; EtBr, ethidium bromide; GFP, green fluorescent protein; PBS, phosphate-buffered saline; scAAV1, self-complementary AAV serotype 1.

Figure 4

Applications of EtBr-enhanced retrograde transduction. (a) NT-3 levels after retrograde transduction with scAAV1-NT-3 are significantly enhanced in lumbar ventral gray matter when viral delivery is preceded by 0.1% EtBr injection (Kruskal–Wallis χ² P < 0.005). (b,c) Sensory neurons retrogradely transduced following sciatic demyelination are visible through the dorsal surface of the live mouse spinal cord in vivo using two-photon microscopy. Bar = 500 µm (b), 100 µm (c). ELISA, enzyme-linked immunosorbent assay; EtBr, ethidium bromide; scAAV1, self-complementary AAV serotype 1.