There is a relative abundance of strategies and methodologies to facilitate drug delivery to the central nervous system. However, drug delivery directly to the peripheral nervous system is less common, with fewer detailed methods publications available to aid researchers. Here, we describe a direct nerve injection method for peripheral nervous system drug delivery, using the vagus nerve as a model nerve. This method can be used in the treatment of autonomic nervous system disorders through targeting of the left vagus nerve, although this general injection method can be extrapolated to injection of other nerves with minor modification. This method explains all critical steps involved in the procedure involving microsurgery in anesthetized adult rats under a dissecting microscope. The use of a tracking dye is described to facilitate the monitoring of injection fidelity in real time. Illustrations of successful and failed injections are provided. If carried out properly, direct vagus nerve injections can be conducted in a safe manner that is well-tolerated by the rat without post-delivery complications. For example, once the surgeons were trained in this method, six out of six rats were successfully injected without any complications. This method of direct nerve injection for preclinical rat studies is capable of delivering agents (including but not limited to gene therapy) to peripheral nerves.

There is a relative abundance of strategies and methodologies to facilitate drug delivery to the central nervous system. However, drug delivery directly to the peripheral nervous system is less common, with fewer detailed methods publications available to aid researchers. Here, we describe a direct nerve injection method for peripheral nervous system drug delivery, using the vagus nerve as a model nerve. This method can be used in the treatment of autonomic nervous system disorders through targeting of the left vagus nerve, although this general injection method can be extrapolated to injection of other nerves with minor modification. This method explains all critical steps involved in the procedure involving microsurgery in anesthetized adult rats under a dissecting microscope. The use of a tracking dye is described to facilitate the monitoring of injection fidelity in real time. Illustrations of successful and failed injections are provided. If carried out properly, direct vagus nerve injections can be conducted in a safe manner that is well-tolerated by the rat without post-delivery complications. For example, once the surgeons were trained in this method, six out of six rats were successfully injected without any complications. This method of direct nerve injection for preclinical rat studies is capable of delivering agents (including but not limited to gene therapy) to peripheral nerves.

Background/Objectives: Direct intraneural delivery of adeno-associated virus serotype 9 (AAV9) via peripheral nerves remains largely unexplored. This study evaluated the feasibility of AAV9 delivery into the vagus nerve, identified an optimal dose, and determined an optimal fluorescein dye concentration for tracking injection fidelity. Methods: Rats received left vagus nerve injections of 5 µL AAV9/GFP at doses of 2.5 × 10⁹, 2.5 × 10¹⁰, 7.6 × 10¹⁰, 2.5 × 10¹¹, or 6.5 × 10¹¹ vector genomes (vg)/rat, mixed with 1% luxol fast blue dye. Controls received vehicle with dye. Transduction in the nodose ganglia of the vagus nerve and medulla was assessed. Fluorescein compatibility was evaluated in CHO Lec2 cells using concentrations of 0%, 0.0001%, 0.004%, and 0.01% at high and low multiplicities of infection. In vivo compatibility was subsequently confirmed using 2.5 × 10¹¹ vg/rat with 0.001%, 0.002%, 0.004%, or 0.008% fluorescein. Results: Transduction of critical autonomic nervous system sites increased with dose. The two highest doses (2.5 × 10¹¹ and 6.5 × 10¹¹ vg/rat) produced abundant, comparable transduction in the nodose ganglia and medulla, identifying 2.5 × 10¹¹ vg/rat as the optimal dose. Fluorescein did not inhibit AAV9 transduction in CHO Lec2 cells at the concentrations tested. In vivo, 0.004% fluorescein provided clear visualization during vagus nerve injection. Conclusions: Direct intraneural administration of AAV9/GFP at 2.5 × 10¹¹ vg/rat with 0.004% fluorescein offers an effective formulation for vagus nerve delivery in rats. Further validation and dose-scaling studies are suggested to establish feasibility for translation to larger animals and humans.