Methods
Animals and surgical methods – All surgical methods were approved by the Institutional Animal Care and Use Committee of Emory University (Protocol No. PROTO201800101). Adult C57B6/J mice of both sexes were used. These animals were considered wild type (WT). Transection and repair of the sciatic nerve was performed as described previously (Akhter et al. , 2019). Briefly, in isoflurane-anesthetized animals, the sciatic nerve was exposed in the thigh, cut and immediately repaired by end-to-end anastomosis, and secured in place using fibrin glue. The glue was prepared at the time of surgery from fibrinogen and thrombin (Akhter et al. , 2019) and nothing was added to the glue. All incisions were then closed and the mice returned to their cages. On the third day following surgery, the mice began daily treatments, five days per week for two weeks, either with CP11 (Santa Cruz Biotechnology, catalog # sc-319780) (10 mg/kg) or vehicle (4% DMSO in sesame oil). In one set of experiments (12 mice: six CP11-treated and six vehicle-treated, three males and three females in each treatment group) treatments were administered via intraperitoneal injection. In an additional cohort of eight mice, the CP11 or vehicle treatments were given orally. Doses administered were chosen based on published results (Zhang et al. , 2017).
Measurement of AEP enzymatic activity – Under isoflurane anesthesia, sciatic nerves were cut and repaired in WT mice, as described above. Beginning on the third post-operative day, mice were given either CP11 (10mg/kg) or vehicle, orally, each day. One group of animals was euthanized with Euthasol® solution (pentobarbital sodium and phenytoin sodium, 150 mg/Kg) three days after the initial treatment. A second set of animals were euthanized after seven days of treatments. The cut and repaired nerves were harvested, including 1 mm proximal and distal to the injury site. Nerves were harvested from a third group of intact mice that served as a control.
The AEP activity assay used was a modification of one previously described (Wang et al. , 2018). Freshly made nerve tissue homogenates (25 μg) in Lysis buffer (50 mM Tris·HCl,pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 5 mM EDTA, 5 mM EGTA, 15 mM MgCl2, 60 mM β-glycerophosphate, 0.1 mM sodium orthovanadate, 0.1 mM NaF, 0.1 mM benzamide, 10 mg/mL aprotinin, 10 mg/mL leupeptin, and 1 mM PMSF) were prepared and incubated with 100 μL reaction buffer (50 mM Sodium Citrated, 0.1% CHAPS, and 1 mM DTT, pH 6.0) containing 10 μM AEP substrate, Z-Ala-Ala-Asn-AMC (Bachem). The AMC released by AEP-mediated substrate cleavage was quantified at 360/460 nm in a fluorescence plate reader at 37 °C in kinetic mode. For quantification, densitometry readings were scaled to the maximum value of all of the specimens tested.
Recording of compound muscle action potentials (M responses) – Four weeks after sciatic nerve transection and repair, and two weeks after the end of treatments, the success of motor axon regeneration and reinnervation to the tibialis anterior (TA) and lateral gastrocnemius (LG) muscles was evaluated. In isoflurane anesthetized animals, the sciatic nerve was exposed as it leaves the pelvis and two needle electrodes (Ambu #74325-36/40, Columbia, MD, United States) were placed in contact with it. Bipolar fine wire (Stablohm 800 A, California Fine Wire) electrodes (Basmajian & Stecko, 1963) were inserted transcutaneously into the centers of the TA and LG muscles using a 25G hypodermic needle. The free ends of the wires were connected to the head stages of differential amplifiers. Ongoing activity recorded from these muscles was sampled at 10 KHz by a laboratory computer system running custom Labview® software and when activity over a 10 ms period was within a user-defined background range, the computer delivered a single brief (0.3 ms) constant voltage pulse to the nerve via the needle electrodes and recorded EMG activity for 50 ms. In each animal, a range of stimulus intensities was applied, extending from subthreshold to supramaximal. To avoid fatigue, stimuli were delivered no more frequently than once every five seconds. Amplitudes of the recorded direct muscle (M) responses were measured as the average full wave rectified voltage between the onset and duration of the recorded triphasic action potential. For each muscle tested in each mouse studied, the amplitude of the largest M response (Mmax) was determined.
Retrograde labeling experiments – The number of motor and sensory (dorsal root ganglion, DRG) neurons that successfully regenerated axons and reinnervated the TA and gastrocnemius (GAST) muscles was investigated using the application of retrograde fluorescent tracer molecules to these muscle targets, four weeks after bilateral sciatic nerve transection and repair, and two weeks after the cessation of daily treatments. This survival time was chosen to be compatible with those of previous studies (Al-Majed et al. , 2000b; Englishet al. , 2009; English et al. , 2011a; English et al. , 2011b; Udina et al. , 2011a; Gordon & English, 2016) evaluating activity-dependent experimental therapies to enhance peripheral axon regeneration. At the end of the electrophysiological experiments described above, the TA and gastrocnemius (GAST) muscles were exposed in the anesthetized animals. Two microliters of a 1% solution of wheat germ agglutinin (WGA), conjugated either to Alexa Fluor 488 (TA) or Alexa Fluor 555 (GAST), was injected into each muscle using a Hamilton microliter syringe equipped with a 36G needle. Small amounts of tracer were injected at two locations in each muscle and the needle was left in place for five minutes between injections to minimize leakage of the tracer along the needle track. After washing the entire surgical field three times with normal saline, surgical wounds were closed in layers before animals were returned to their cages. Three days later, the mice were euthanized by intraperitoneal injection of Euthasol and perfused transcardially with saline followed by 4% paraformaldehyde, pH 6.9. Lumbar spinal cords and L4 dorsal root ganglia (DRGs) were harvested and cryoprotected for at least 24 hours in 20% sucrose solution. Cryostat sections of spinal cords, in a horizontal plane at 40 µm thickness, were mounted onto charged slides and cover slipped using Vectashield®. Images of these sections at 20X magnification, using a Leica DM6000 upright fluorescence microscope, HC PL APO 0.70 NA objective, and Hamamatsu low-light camera, were made using HCImage software. Labeled motoneurons were identified if the retrograde fluorescence filled the soma and extended into the proximal dendrites and if a clear area of the cell corresponding to the nucleus could be visualized, as described previously (English, 2005). Profiles of motoneurons that did not meet these criteria were not counted. Harvested dorsal root ganglia were sectioned on a cryostat at 40 µm thickness, mounted onto charged slides and cover slipped using Vectashield®. Imaging of these sections was identical to that used for spinal cords, above. A DRG neuron was considered labeled if the fluorescent marker filled the entire soma and a clear nuclear region could be identified.
Cell cultures – Mouse lumbar dorsal root ganglion cells were harvested to assay neurite outgrowth after drug treatment. Mice were decapitated under isoflurane anesthesia. The entire vertebral column was removed and immediately placed on a cooled surface under a laminar flow hood. Individual ganglia (L1-L6) were dissected and pooled in a tube containing room temperature Hanks’ Balanced Salt Solution (HBSS). Following ganglia collection, the HBSS was removed and a dispase-collagenase solution was added back to the tube, which was then placed in a 37˚C bead bath for an hour-long incubation. During this one-hour period, the tube containing ganglia was briefly removed from the bead bath every 10 minutes and gently agitated by hand to ensure even enzymatic digestion of tissue. After incubation, dispase-collagenase was removed and replaced with DNase for 2.5 minutes. Then, without removing the DNase, pre-warmed (37˚C) HBSS was added to the tube and tissue was further dissociated into a cell suspension by repeatedly pipetting with a P1000 pipet. The cell suspension was centrifuged at 1000 rcf for 3 minutes. The supernatant was subsequently discarded and the remaining pellet of cells was resuspended in Neurobasal A medium supplemented with B-27 (2%), GlutaMAX (1%), and penicillin-streptomycin (1%). Cells (3000/coverslip) were seeded directly onto laminin- and poly-D-lysine-coated 12 mm glass coverslips placed at the bottom of each well of a 4-well plate and the volume of media in each well was brought to 500 µL. Plates were stored in a water-jacketed incubator maintained at 37˚C and steadily supplied with 5% CO2. Twenty-four hours after plating, half of the media from each well was removed and replaced with fresh media with or without the drugs of interest. After allowing an additional 24 hours of incubation, cells were fixed in a solution of 4% paraformaldehyde in phosphate-buffered saline (PBS), then washed three times for five minutes in cold PBS, and stored in PBS at 4˚C for up to a week before immunofluorescent antibody detection. Cultures used cells derived from both WT mice and AEP knockout (KO) mice (Shirahama-Noda et al. , 2003). All mice used were genotyped from tail samples by Transnetyx, Inc. prior to use.
Immunofluorescence Staining of Cultured Neurons – Paraformaldehyde-fixed dorsal root ganglion cells attached to 12 mm glass coverslips were incubated with primary antibodies to protein targets (Table I), and then fluorescent secondary antibodies for detection (Table I). After blocking nonspecific binding and permeabilizing cells with a buffer (10% donkey serum and 0.3% Triton X-100 in PBS) at room temperature for one hour, primary antibodies were diluted in this same buffer, and the plate containing the cells was kept in continuous agitation at 4˚C overnight. The following morning, cells were washed in PBS and then incubated with secondary antibodies in buffer at room temperature for two hours before washing again and mounting on glass slides with Vectashield with DAPI. Images of cells were captured at 20X magnification as described above. Neurite lengths were measured using the FIJI software package.
Statistical analyses – Numbers of animals in experimental groupings used were deemed adequate based on a post-hoc power analysis performed using G*Power 3.1 (Power = (1-β err prob) >0.8). All statistical comparisons were performed using GraphPad Prism software. If results of analyses of variance were significant,post-hoc paired testing using the two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli was employed, unless noted otherwise. Alpha for significance of differences was set at p<0.05 throughout.