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SUPPLEMENTAL METHODS
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Nerve-crush injury
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Nerve-crush injury was induced as described previously.1 Briefly, mice were anesthetized via
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isoflurane inhalation, then the sciatic nerve was exposed at mid-thigh level and crushed for 15
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seconds with a hemostat; the incision was closed, and mice were kept on a heating plate at 37°C
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until fully recovered from the anesthesia. Analgesia was provided with intraperitoneal injections
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of buprenorphine immediately after surgery and with orally administered meloxicam solution for
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up to 3 days after surgery.
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Motor-nerve conduction velocity (MCV)
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Measurements were performed in sedated mice at the level of the sciatic-peroneal nerves by
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using standard orthodromic surface recording techniques and a TECA TD-10 portable recording
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system (Oxford Instruments, Pleasantville, NY, USA). Electrodes were inserted at the sciatic
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notch and the Achilles tendon, and electromyograms were recorded in the interosseus muscles of
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the foot. Compound muscle action potentials (CMAPs) were monitored by stimulating the
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peroneal nerve at the posterolateral ankle or by stimulating the sciatic nerve percutaneously with
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a monopolar needle electrode; the latency difference between the peaks of the CMAPs evoked by
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stimulation at the two sites was determined, then MCV was calculated by dividing the distance
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between the stimulating electrodes by the average difference in latency. To correct for potential
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differences in ambient light, temperature, body temperature, sedation, and other confounding
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factors, measurements in the injured limb were normalized to measurements performed in the
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uninjured, contralateral limb.
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Duration of rotarod exercise
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In the rotarod test,2 mice were placed on the rotarod cylinder, the rotational speed was increased
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from 4 rpm to 40 rpm over 10 minutes, and the duration of exercise (i.e., the length of time until
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the animal fell off the cylinder or gripped the cylinder and spun for two revolutions) was
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recorded; the maximum of 3 measurements was reported for each mouse. Mice were trained 6
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times over a two week period before experimental data was acquired.
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qRT-PCR
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RNA was extracted from homogenized nerve tissue or from 5×105 cells with RNA STAT-60
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(TEL-TEST, Inc., Friendswood, TX, USA) as directed by the manufacturer’s instructions. Total
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RNA was reverse transcribed with a Taqman cDNA Synthesis Kit (Applied Biosystems, Foster
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City, CA, USA) and amplified with the SDS 7500 FAST Real-time PCR system (Applied
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Biosystems); primer and probe sequences are provided in Supplemental Table. The PCR
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procedure consisted of a 2-minute hold at 50°C, and then a 10-minute hold at 95°C followed by
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40 2-step cycles between 95°C for 15 seconds and 60°C for 60 seconds. Relative mRNA
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expression was calculated with the comparative CT method (relative expression=2ΔCT) and
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normalized to the expression of the endogenous 18S gene.
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Gli-luciferase assay
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HUVECs were transfected with Renilla luciferase (pRL-TK, Promega Corporation, Madison,
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WI, USA) and with a Gli-luciferase reporter construct by using Fugene6 (F. Hoffmann-La Roche
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Ltd, Basel, Switzerland) as previously described3 and cultured for 24 hours; then, the medium
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was replaced with low-serum medium (0.5% FBS/EBM or FBS/DMEM), and the cells were
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cultured for another 8 hours. Cells were treated with E2 (7×10-8M) for 6 hours and with or
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without 1 µg/mL Shh for 18 hours; then, luciferase activity was assayed with a luciferase-assay
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system (Promega Corporation), and normalized to the Renilla luciferase control to compensate
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for differences in transfection efficiency.
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SUPPLEMENTAL REFERENCES
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1.
De Koning P, Brakkee JH, Gispen WH. Methods for producing a reproducible crush in
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the sciatic and tibial nerve of the rat and rapid and precise testing of return of sensory
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function. Beneficial effects of melanocortins. J Neurol Sci 1986;74:237-246.
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2.
Jeong SW, Chu K, Jung KH, et al. Human neural stem cell transplantation promotes
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functional recovery in rats with experimental intracerebral hemorrhage. Stroke
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2003;34:2258-2263.
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3.
Pola R, Ling LE, Aprahamian TR, et al. Postnatal recapitulation of embryonic hedgehog
pathway in response to skeletal muscle ischemia. Circulation 2003;108:479-485.
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Supplemental Table. Primers and probes used for real-time qRT-PCR analyses.
Forward (F)/Reverse(R) primer
Probe
F: 5’-CGGGTCGGGAGTGGGT-3’
5’- Cal Fluor
R: 5’-GAAACGGCTACCACATCCAAG-3’
Orange-TTTGCGCGCCTGCTGCCTT-BHQ-3’
18S
F: 5’-CAGCGACTTCCTCACCTTCCT-3’
5’- Fam-ACCGCGACGAAGGCGCCA-BHQ-3’
Shh
R: 5’-AGCGTCTCGATCACGTAGAAGAC-3’
F: 5’- GCTTGGATGAAGGACCTTGTG-3’
5’- Fam-ACTCTCCACGCTTCGCCGCCT-BHQ-3’
Gli1
R: 5’- GCTGATCCAGCCTAAGGTTCTC-3
F: 5’-TGTGGTCATCCTGATTGCATCT-3’
5’-
R: 5’-GTCCCCAATGGCTGTCAGA-3’
Fam-ACCGTCCACGTGGCTTTGGCCT-BHQ-3’
Ptch1
F: 5’- GCAGGCTGCTGTAACGATGA-3’
5’-FAM-CCCTGGAGTGCGTGCCCACG-BHQ-3’
VEGFA
R: 5’-GCATGATCTGCATGGTGATGTT-3’
F: 5’-GAATGCAGAGTCACAGTTCAACCT-3’
5’-FAM-CCCAGCCACTGACCTCCGATTGC-BHQ
R: 5’-GGTGTAGTAGCCGTTTCGACAGA-3’
-3’
HHIP
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Supplemental Figure 1
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Local E2 injection does not significantly increase serum E2 levels. One week before surgical
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sciatic nerve-crush injury, E2 (100 μg) in poly lactide-CO glycoside (PLGA) (to ensure extended
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E2 delivery) or PLGA alone (placebo) was locally injected into the designated injury site, or an
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extended-release E2 pellet (0.5 mg delivered over 60 days) was subcutaneously implanted into
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the limb. Serum E2 levels were evaluated 1-28 days after injury.
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Supplemental Figure 2
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E2 does not alter Ptch1 or Gli1 expression in cultured endothelial cells, fibroblasts, or Schwann
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cells. (A) Ptch1 and (B) Gli1 mRNA expression was evaluated in HUVECs, fibroblasts (NIH
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3T3), and Schwann cells (SW10) that had been treated with 1×10–8 mol/L E2 for 6 hours;
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measurements were performed via qRT-PCR and normalized to endogenous 18S rRNA levels.
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ND indicates not detectable.
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