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.