AAV vector production, purification, and titration. For all transgene expression studies with AAV vectors we used the AAV expression plasmid, pAAV-CAG-tdTomato (codon diversified), which was a gift from Edward Boyden (Addgene plasmid # 59462; http://n2t.net/addgene:59462). This plasmid contains AAV inverted terminal repeats (ITRs) flanking the tdTomato expression cassette which consists of: a chicken β-actin (CAG) promoter, Kozak sequence, tdTomato cDNA, woodchuck hepatitis virus post transcriptional regulatory element (WPRE), and an SV40 poly A signal sequence. We packaged this AAV transgene expression cassette into the following capsids: (1) AAV-F (AAV-F in pAR-9 was a gift from Casey Maguire, Addgene plasmid # 166921; http://n2t.net/addgene:166921); (2) AAV9 (pAR9 plasmid obtained from the Massachusetts General Hospital virus vector core); and (3) AAV6 (plasmid pAAV-RC6, Cell Biolabs, Inc, San Diego, CA) [32]. AAV production of AAV-F, AAV9, and AAV6 was performed as previously described [82]. Briefly, 293T cells were triple transfected (calcium phosphate method) with (1) AAV rep/cap plasmid (2) an adenovirus helper plasmid, pAdΔF6, and (3) pAAV-CAG-tdTomato. Cell lysates were harvested 68–72 hours post transfection and purified by ultracentrifugation of an iodixanol density gradient. Iodixanol was removed and buffer exchanged to phosphate buffered saline (PBS) using Zeba desalting columns, 7 kDa molecular weight cutoff (MWCO; Thermo). Vector was concentrated from 4 ml to approximately 1 ml using 2 ml Amicon Ultra 100 kDa MWCO ultrafiltration devices. We obtained PHP.S vector packaging the same AAV-CAG-tdTomato cassette from Addgene (Catalog # 59462-PHP.S, Viviana Gradinaru laboratory) [33]. Addgene also uses iodixanol gradient purification. Vector titers in VG/ml were determined by Taqman qPCR in an ABI Fast 7500 Real-time PCR system (Applied Biosystems) using probes and primers to the AAV ITRs and an AAV plasmid standard curve. We titered all four vectors (AAV-F, PHP.S, AAV9, and AAV6) on the same qPCR run to ensure accuracy of the head-to-head comparison experiments. Vectors were pipetted into single-use aliquots and stored at -80°C until use.
Conduit reservoir delivery of AAV-CAG-tdTomato and Fluoro-Gold to Transected Facial Nerves. Twenty-four adult C57 mice (7–9 weeks, 12 female) were used for vector and Fluoro-Gold™ (FG) (Fluorochrome LLC, Denver, Colorado) delivery. All animal surgeries were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and with approval by the Massachusetts Eye and Ear Animal Care Committee (ACC Protocol # 16 − 006, IRBNet ID 884247). This study was carried out in compliance with the ARRIVE guidelines.
Procedures were performed under isoflurane anesthesia, 1–3% maintenance dosing in 1 L/min O2. Buprenorphine (0.05 mg/kg subcutaneously) and meloxicam (1.0 mg/kg subcutaneously) were administered prior to skin incision. A 1 cm infra-auricular incision was made on the left, and skin-muscle flaps were elevated. The exorbital lacrimal gland was retracted to expose the buccal branch of the facial nerve. This branch was meticulously dissected circumferentially using the operating microscope at 25x magnification. Care was taken to avoid crush injury. The nerve was transected just proximal to the distal pes and the proximal stump then immersed in a pipette tip containing 5 µL of titer-matched vector solution (n = 4 mice/vector group, 2 males and 2 females per vector group) (5.9x1011 VG/mL) for 10 minutes (Supplementary Figs. S1 and S2a). In four control mice, the proximal nerve stump was dipped in 5 uL of 2% FG (Fluorochrome, Denver, CO, w/v in distilled water) for 10 minutes. In a second control group of three mice, the nerve was transected but no virus or dye was delivered. In one experiment, high-dose AAV-PHP.S (1.72x1013 GC/mL) carrying the pAAV-CAG-tdTomato transgene expression cassette was delivered to one mouse nerve. After nerve transection, the proximal stump was placed overlying the masseteric fascia, separated from its distal stump. The wound bed was irrigated with saline and closed in a single layer using 4 − 0 absorbable suture (Polysyn, Sharpoint, Westwood, MA). Animals recovered from general anesthesia and returned to their cages. Postoperative Meloxicam was given for 72 hours post-procedure.
Three weeks after AAV delivery, the 16 vector-treated mice (n = 4 mice/vector x 4 vectors) and three mice who had undergone prior nerve transection without delivery of virus, underwent isoflurane anesthesia at the above dosing with the same analgesics. A 1 cm infra-auricular incision was made on the left and the previous buccal branch transection was identified. The nerve was transected just proximal to the neuroma and the proximal stump immersed in 5 µL of 2% FG (Supplementary Fig. S9b). The high-dose AAV-PHP.S mouse underwent facial nerve main trunk transection with proximal stump immersion in 5 µL of 2% FG. The wound bed was irrigated with saline and closed in a single layer using 4 − 0 absorbable suture (Polysyn). Animals were recovered from general anesthesia. Postoperative Meloxicam was given for 72 hours post-procedure.
Tissue harvest. Six days following FG delivery, animals underwent CO2 euthanasia and cardiac perfusion using 2% phosphate-buffered paraformaldehyde fixative (PFA) solution. Animal heads were placed in 2% PFA overnight, then underwent brainstem harvest at the level of the facial nucleus. The intracranial facial nerve was used as a landmark for facial motor nucleus identification. Brainstems were placed in PBS in a light-tight container and stored at 4o C for seven days prior to whole mount imaging.
Immunofluorescence staining of brain stems.
One animal from each treatment group underwent CO2 euthanasia followed by cardiac perfusion using 4% PFA solution. Animal heads were placed in 4% PFA for 48 hours prior to brainstem harvest and overnight cryoprotection in 30% sucrose solution. Brainstems were then embedded in O.C.T. media (Tissue-Tek) and cryosectioned in the coronal plane at 40 µm. Floating sections were permeabilized with 0.5% Triton X-100 in PBS for 30 minutes at room temperature and blocked with 5% normal chicken serum (NCS, Abcam) in PBS for 1 hour at room temperature. Primary antibodies were incubated overnight (1:200 dilution factor) at 4°C in 1.5% NCS PBS. Primary antibodies used for this study were: rabbit anti-red fluorescent protein (RFP, Code: 600-401-379, Rockland Antibodies,.); goat anti-ChAT (cat#AB144P EMD Millipore, Burlington, USA).
After washing sections, fluorophore-conjugated secondary antibodies were incubated in 1.5% NCS in PBS at 1:1000 dilution. The secondary antibody for anti-RFP primary was chicken anti-rabbit Alexa Fluor 594 (Thermo, Cat A-21442) and the secondary antibody for anti-ChAT primary was chicken anti-goat Alexa Fluor 488 (Thermo, Cat A-21467). After washing in PBS, nuclei were labeled with a 1:10,000 dilution of 4′,6-diamidino-2-phenylindole (DAPI). Sections were mounted onto slides with a fine brush, dried for 2 hours, and cover slipped with Dako fluorescent mounting medium (Agilent).
Imaging of whole mount brainstems and immunofluorescent tissue sections.
For IF imaging, sections were mounted with immersion oil (Code 1261, Cargille Laboratories, Cedar Grove, NJ) and imaged at multiple focal planes using a 40x objective (0.24mm WD, HC PL APO 40x/1.3 Oil CS2, Leica) on a Leica DM 6000 CS confocal microscope. For Alexa Fluor 488 visualization, an Argon excitation laser was used and emitted fluorescence between 500–550 nm was collected. For Alexa Fluor 594 visualization, a Diode-pumped solid state (DPSS) 561 laser was used for excitation and emitted fluorescence between 600–650 nm was captured.
2PEM imaging was performed on a commercial multiphoton microscope (TrimScope II, LaVision Biotech) powered by a dual-output femtosecond laser (Insight X3, SpectraPhysics) at 830 nm and 1045 nm. Images were acquired with a set of galvanometer mirrors and piezo XYZ-stage for large-field volumetric imaging. Commercial image-analysis software (Bitplane Imaris 9.2; Oxford Instruments, Zurich, Switzerland) was used for stitching of tile scan 2PEM images. Optical clearing was achieved using a glycerol-immersion objective lens (CLr Plan-Neofluar 20x, Carl Zeiss) and refractive index matching solution (EasyIndex, LifeCanvas Technologies, Cambridge, MA). Fluorescent signal was spectrally filtered, a combination of short-pass and long-pass filters were used for collecting below 495 nm in the FG channel and above 560 nm in the tdTomato channel.
Facial motor neuron cell body counts were quantified from original 3D data sets using commercial machine learning software (Aivia v9.5, Leica Microsystems, Bellevue, WA). A random forest pixel classifier was trained by painting examples of cell body signal and background signal [83]. This classifier was used to generate a signal channel in Aivia’s 3D object mesh recipe console to highlight and segment neuronal cell bodies. The object meshes were generated in a region of interest and adjusted in an iterative manner, during which morphological smoothing was performed, minimum object radius and edge intensity defined, and holes filled until a satisfactory result was obtained. The segmentation parameters were then applied to the entire volume to generate a channel highlighting the pixels comprising cell bodies and automated cell counts were performed. This process was performed independently for FG-labeled and tdT-expressing cell bodies. The segmentation parameters were kept consistent for all reconstructions and all analyses were blinded to vector.
Statistical analyses
All statistical analyses were performed using SPSS (IBM SPSS Statistics 27). Levene’s test was used to verify homogeneity of variances. Normality of data was confirmed using the Shapiro-Wilk and Kolmogorov-Smirnov tests. One-way AVOVA followed by Bonferroni post hoc analysis was performed to compare cell bodies expressing tdT, as well as FG-labeling of neuronal soma. One-way ANOVA with Games-Howell post hoc analysis was used to compare surface area and volume of segmented cell bodies. Confidence level was assessed at 95% (P < 0.05).