Mice were raised in a barrier facility in the Hill Pavilion, University of Pennsylvania. All animal procedures were conducted according to animal protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Pennsylvania. For in-situ hybridization and antibody characterization studies, six P14-P21 wild type mice of both sexes with a mixed CD1/C57Bl/6 J background were used. For RT-PCR experiments, four 4–6 month wild type mice of both sexes with a mixed CD1/C57Bl/6 J background were used. For dorsal rhizotomy experiments, we used 2 month old C57Bl/6 J mice of either sex and the survival surgery was conducted in Shriners Hospitals Pediatric Research Center, Temple University. All surgical and postoperative procedures were performed in accordance with Temple’s Institutional Animal Care and Use Committee and National Institutes of Health guidelines.
In situ hybridization
DIG-labeled riboprobes were synthesized using a DIG RNA labeling kit (Roche, 11175025910). Template for Nmb probe was amplified from a mouse DRG cDNA library (BD, Ref#630022) and subcloned into vector pGEM-T Easy (Promega, A1360). Mouse IMAGE clones for Grp (GenBank: BC024515), Grpr (GenBank: BC113145), and Nmbr (GenBank: BC119237) were purchased from Open Biosystems, and PCR products were subcloned into pGEM-T Easy. Primers used to amplify cDNA were: Nmb (5’-GGCAAGCAGGGAGCTCTT-3’ and 5’-CTGGTGACCCAACCAGAA-3’), Grp (5’-CACGGTCCTGGCTAAGATGTAT-3’ and 5’-CCAGTAGAGTTGACGTTTGCAGA-3’), Nmbr (5’-AGGTCTCTCTCCAACCTCTCCT-3’ and 5’-ACCAGAACAATCTTAGCCAGGCG-3’), and Grpr (5’-ATGGCTCCAAATAATTGTTCCCA-3’ and 5’-TTTAGTCTAGACATACCCCTCAT-3’). FITC-labeled probes for c-Ret, TrkB, TrkC, TrpV1, MrgprA3, MrgprD, and TrpM8 were generated as previously described [23, 35].
Intact lumbar spinal column was dissected from euthanized wild-type P14-P21 mice and rapidly frozen in OCT on a dry-ice/ethanol bath. 20 μm cryosections were collected on Superfrost Plus slides (Fisher, 22-034-979) and allowed to dry for at least 2 hours at room temperature before in-situ hybridization. All steps prior to hybridization were carried out under RNase free conditions. Cryosections were immersion-fixed in freshly made 4% PFA in PBS for 20 minutes at room temperature. Slides were then washed in fresh-DEPC PBS (1:1000 DEPC in PBS immediately before use), followed by wash in DEPC-pretreated PBS (1:1000 DEPC in PBS overnight (O/N), followed by autoclaving). An antigen retrieval step, often used for immunohistochemistry, was found to increase the signal of many probes. Citric acid buffer (10 mM citric acid, 0.05% Tween-20, pH6.0) was boiled in a microwave, and DEPC (1:1000) was added to freshly boiled solution. Slides were immersed in solution in a 95°C waterbath for 20 minutes, and then allowed to cool at room temperature for 30 minutes. Sections were then washed in DEPC pretreated PBS (1X 5 minutes), incubated in Proteinase K (25 μg/mL in DEPC-pretreated H2O) for five minutes, followed by washes in fresh-DEPC PBS (1X 5 minutes) and DEPC pre-treated PBS (1X 5 minutes). Sections were then acetylated at room temperature for ten minutes in freshly made acetylation solution (0.1 M triethanolamine, 0.25% acetic anhydride in DEPC pre-treated H2O). Slides were then prehybridized in hybridization buffer (50% formamide, 5XSSC, 0.3 mg/mL yeast tRNA, 100 μg/mL heparin, 1X Denhardt’s, 0.1% Tween-20, 0.1% CHAPS, 5 mM EDTA in RNase free H2O) at 62°C in a humidified chamber for 30 minutes. Following prehybridization, excess hybridization buffer was removed from slides and 1-2 ng/μl of DIG and/or FITC labeled riboprobe diluted in hybridization buffer was placed on the slide. Slides were incubated O/N under Parafilm coverslips at 62°C. Slides were then washed in 0.2X SSC at 68°C (1X 15 minutes, 2X 30 minutes).
For colorimetric reaction, slides were blocked in PBT (PBS, 0.1% TritonX-100) and 20% lamb serum at room temperature for one hour. Sections were then incubated with AP-conjugated anti-DIG antibody (1:1000; Roche, 11093274910) in blocking buffer O/N at 4°C. Slides were washed in PBT (3X 10 minutes) and incubated O/N in darkness in alkaline phosphatase buffer (100 mM Tris pH9.5, 50 mM MgCl2, 100 mM NaCl, 0.1% Tween-20, 5 mM levamisole, 0.34 mg/mL 4-Nitro blue tetrazolium (NBT)(Roche, 11383213001), 0.17 mg/mL 5-bromo-4-chloro-3-indolyl-phosphate(BCIP)(Roche, 1138221001)). Following color reaction, slides were rinsed repeatedly in PBS and then fixed for 20 minutes in 4% PFA in PBS at room temperature. Slides were then repeatedly rinsed in ddH2O, dried at 37°C for 1 hour, dehydrated in xylenes (3X 2 minutes), and coverslipped with Permount (Fisher, SP15).
For double fluorescent in-situ hybridization (FISH), slides were blocked for one hour at room temperature with 0.5% Blocking Reagent (Roche, 11096176001) in PBS. Sections were incubated in anti-FITC-POD (1:100 in .5% Blocking Reagent; Roche, 11426346910) O/N at 4°C. Slides were then washed in PBT (3X10 minutes) and incubated in 0.1% BSA in PBS for 15 minutes. FITC riboprobes were then developed using the TSA Plus system (Perkin Elmer, NEL741001KT), by diluting fluorescein tyramide into 1X amplification buffer (1:100) and incubating slides in working solution for 10–15 minutes, followed by washes in PBS (3X 10 minutes). Slides were then blocked in PBT containing 20% lamb serum for 1 hour at room temperature, and incubated O/N at 4°C with AP-conjugated anti-DIG antibody (1:500 in PBT +20% lamb serum). Slides were washed in TNT (100 mM Tris–HCl, 150 mM NaCl, 0.05% Tween-20, pH7.5) (3X 10 minutes), then in detection buffer (100 mM Tris–HCl, 100 mM NaCl, 10 mM MgCl2, pH8.0) (2X 10 minutes). DIG-labeled riboprobes were then developed using HNPP/Fast Red TR system (Roche, 11758888001). Sections were incubated in detection solution (10μL HNPP stock solution, 10μL of 25 mg/mL FastRed per 1 mL of detection buffer, filtered through 0.2 μM nylon filter) (3x30 minutes), with TNT rinses between incubations. Slides were then rinsed in PBS and mounted with Flourmount (Southern Biotech, 0100–01).
For FISH combined with immunofluorescence, normal hybridization procedure was followed, using DIG-labeled probe. After 0.2X SSC washes, sections were blocked for one hour in PBT containing 20% lamb serum. Sections were then incubated with AP-conjugated anti-DIG (1:500) and primary antibody at the appropriate dilution (described below) at 4°C O/N in 20% lamb serum blocking solution. Slides were washed in PBT (3X 10 minutes), then incubated in species appropriate Alexa 488 conjugated secondary antibody (1:500 in 5% lamb serum in PBT) for one hour at RT. HNPP/FastRed detection was then performed as described above, beginning with initial TNT washes.
For characterization of anti-bombesin antibody, P14-P21 mice were deeply anesthetized with CO2 and perfused with 4% PFA in PBS. Intact lumbar spinal columns were dissected and post-fixed for 2–4 hours in 4% PFA in PBS at 4°C, cryoprotected in 30% sucrose in PBS O/N at 4°C, and embedded in OCT. 20 μm cryosections of lumbar spinal cord and DRG were collected on Superfrost Plus slides, and allowed to dry at room temperature for at least two hours. For dorsal rhizotomy samples, 30 μm free floating cryosections were collected in PBT and processed for immunohistochemistry in solution. Sections were washed in PBT (3X 10 minutes), and then blocked in PBS containing 5% lamb serum and 0.3% TritonX-100 for 1 hour at room temperature. Primary antibodies were diluted in the same buffer, and incubated O/N at 4°C, then washed in PBT (3X 10 minutes). Secondary antibodies were incubated in blocking buffer at 1:1000 dilution for one hour at room temperature. Slides were then washed in PBT (3X 10 minutes) and mounted with Flourmount. Primary antibodies used include rabbit anti-bombesin (1:1000; ImmunoStar, 20073), chicken anti-Gfp (1:2000; Aves, GFP-1020), guinea pig anti-Cgrp (1:250; Bachem, T-5053), mouse anti-Pkcγ (1:50, Invitrogen, 13–3800), chicken anti-Pap (1:1000; Aves, PAP), rabbit anti-peripherin (1:1000; Millipore, AB1530), rabbit anti-Nfh (1:2000; Sigma, N4142), rabbit anti-TrkA (1:1000; Millipore, 06–574), rabbit anti-tyrosine hydroxylase (1:100; Millipore, AB152), mouse anti-NeuN (1:1000, Millipore, MAB377), and Alexa 488 conjugated IB4 (1:200; Invitrogen, I21411). Secondary antibodies used were Alexa 488, Alexa 594, or Alexa 647 conjugated goat anti-rabbit antibody, Alexa 488 conjugated goat anti-mouse antibody, Alexa 488 conjugated goat anti-chicken antibody, and Alexa 488 conjugated goat anti-guinea pig antibody. All secondary antibodies were purchased from Invitrogen.
Anti-bombesin and anti-Cgrp antibodies at the concentrations described above were added to PBS containing 5% lamb serum, 0.3% TritonX-100, and bombesin peptide (50 μg/mL; American Peptide Company, 16-7-10A) or GRP peptide (50 μg/mL; American Peptide Company, 62-3-10) and incubated O/N at 4°C with gentle agitation. Solutions were centrifuged at high speed (~16,000 x g) for ten minutes, and supernatant was used for immunohistochemistry, as described above.
Reverse transcriptase PCR
4–6 month old wild type mice were deeply anesthetized with CO2, transcardially perfused with sterile, RNAse free, ice-cold PBS, then decapitated. Lumbar and thoracic DRGs and dorsal spinal cord were dissected under RNase free conditions and rapidly frozen on dry ice. RNA was isolated using the GeneJet RNA Purification Kit (Fermentas, K0731), and cDNA was synthesized with oligo-dt primers using the SuperScript First-Strand Synthesis system (Invitrogen, 11904018). PCR was performed on cDNA synthesized from DRG or dorsal spinal cord cDNA with primers for Nmb (5’- CCGAGGGACCAGAGACTACA-3’ and 5’-ACTTCACCAGGGAAGCAAGA), Grp (5’-CACGGTCCTGGCTAAGATGTAT-3’ and 5’-CCAGTAGAGTTGACGTTTGCAGA-3’), Nmbr (5’-AGGTCTCTCTCCAACCTCTCCT-3’ and 5’-ACCAGAACAATCTTAGCCAGGCG-3’), Grpr (5’-ATGGCTCCAAATAATTGTTCCCA-3’ and 5’-TTTAGTCTAGACATACCCCTCAT-3’), and Gapdh (5’-GGTGAAGGTCGGTGTGAACG-3’ and 5’-CTCGCTCCTGGAAGATGGTG-3’).
Real time PCR
Grp mRNA from DRG or dSC were measured by quantitative real-time PCR under the ABI 7500 Fast Real-Time PCR system (Applied Biosystems, Foster City, CA), with Gapdh as an internal reference gene. The reaction mixture contained 250 nM of each primer, 1 × SYBR Green PCR Master Mix (ABI), and 1 μl of cDNA. Relative concentration of Grp present in each cDNA sample was calculated by the comparative C(T) method . Real time PCR was performed on cDNA isolated from three adult wild type mice, and was repeated four times per sample. Primers used were Grp primers listed above, and Gapdh (5’-TCGGTGTGAACGGATTTGGC-3’and 5’-TCCCATTCTCGGCTTGACT-3’).
Plasmids were constructed to express bombesin, Grp, or Nmb fused to the C terminus of EGFP. The coding regions of the peptides were constructed by annealing oligonucleotides coding for the open reading frame of each peptide with EcoRI (5’) and SalI (3’) sticky ends. Primers used for this procedure were: bombesin (5-AATTCCAGCAGAGGCTGGGGAATCAGTGGGCAGTGGGTCACTTGATGTGAG-3’ and 5’-TCGACTCACATCAAGTGACCCACTGCCCACTGATTCCCCAGCCTCTGCTGG-3’) Grp (5’-AATTCATGTATCCGCGCGGCAGTCACTGGGCTGTGGGACACTTAATGTGAG-3’ and 5’-TCGACTCACATTAAGTGTCCCACAGCCCAGTGACTGCCGCGCGGATACATG-3’) and Nmb (5’AATTCGGCAACCTCTGGGCGACCGGTCACTTCATGTGAG-3’ and 5’-TCGACTCACATGAAGTGACCGGTCGCCCAGAGGTTGCCG-3’). Oligonucleotides were phosphorylated with T4 polynucleotide kinase (PNK) (NEB, M0201) for 20 minutes at 37°C, and then PNK was heat inactivated at 65°C for 20 minutes. Phosphorylated oligonucleotides were annealed by heating to 95°C for five minutes, then allowing them to cool to room temperature. Annealed oligonucleotides were then ligated into pPMS93 (a gift from Dr. Jeremy Nathans lab at Johns Hopkins University, CMV promoter to drive the expression of EGFP C-terminal fusion protein) pre-digested with EcoRI and SalI. Ligated plasmids were transformed into DH5-α, and grown overnight at 37°C on agarose plates supplemented with 100 μg/mL carbenicillin. Individual colonies were then selected and grown overnight in 2XYT containing 100 μg/mL carbenicillin, and then mini-prepped (Fermentas, K0503). Correct insertions were confirmed by DNA sequencing.
Cell culture and immunocytochemistry
QBI HEK293 cells were grown on 12 mm circle coverslips in HEK293 growth media (10% FBS (Invitrogen, 10082147), 1% Penicillin/Streptomycin (Invitrogen, 15140122) in DMEM (Invitrogen, 11965084)) in 6-well tissue culture plates. Coverslips were coated with 100 μg/mL poly-D-lysine solution to help cell adhesion. Individual cultures were transfected with 1 μg/mL of pRK5-EGFP, pPMS-bombesin, pPMS-Grp, or pPMS-Nmb EGFP fusion plasmid with Lipofectamine LTX (Invitrogen, 15338100). Eighteen to twenty four hours after transfection, coverslips were then rinsed gently with PBS and fixed in 4% PFA in PBS for 1.5 hours at room temperature. Coverslips were then processed for immunohistochemistry as described above.
Dorsal root rhizotomy and postoperative procedures
The surgical procedure was standard . Mice were anesthetized with an intraperitoneal injection of xylazine (8 mg/kg) and ketamine (120 mg/kg). Supplements were given during the procedure as necessary. A 2- to 3-cm-long incision was made in the skin of the back; the spinal musculature was reflected; and the L5 spinal cord segments were exposed by hemi-laminectomies. The cavity made by the laminectomies was perfused with warm sterile Ringer's solution. A small incision was made in the dura overlying the L3-L5 dorsal roots; a fine spring scissor (501778, World Precision Instruments) was introduced subdurally and L4, L5, and L6 dorsal roots were cut. The laminectomy site was covered with a piece of thin synthetic matrix membrane (Biobrane, Bertek Pharmaceuticals), which was then stabilized with a layer of thicker artificial dura (Gore Preclude MVP Dura Substitute, W.L. Gore and Associates). Animals are given subcutaneous injections of lactated Ringer's solution to prevent dehydration and are kept on a heating pad until fully recovered from anesthesia. Buprenex is given as post-operative analgesia (0.05 mg/kg S.C. every 12 hours for 2 days). On the 15th day after dorsal rhizotomy, mice were perfused transcardially with 0.9% heparinized saline solution followed by 4% paraformaldehyde in PBS.
All images were acquired on a Leica DM5000B or Leica TCS SP5 II and processed using Adobe Photoshop CCS version 12.0.
Quantification and statistics
For dorsal rhizotomy experiments, adjacent sections of the spinal cord lumbar enlargement were used for different marker staining. Images of each dorsal horn of individual sections were acquired using identical exposure conditions. Using ImageJ, the bombesin reactive and Cgrp, Pkcγ, or Pap (costain) reactive regions of dorsal spinal cord were outlined. Pixel counts for each fluorescence intensity value (0–255) within the outlined region were generated by ImageJ. In addition, background pixel intensity values were generated from a non-reactive area of the dorsal spinal cord for each image. Background pixel counts were clustered around low intensity values and showed a normal distribution, and the threshold for background fluorescence cutoff was established as the first highest intensity value that had a pixel count of zero. To calculate total fluorescence intensity, the pixel count for each intensity above background level was multiplied by its intensity, and the results of these calculations were summed. In cases where the immunostaining signal was greatly reduced on transected side, the stain area was approximated based on the control side. The difference between the control and transected side was calculated as percentage change in fluorescent intensity ([fluorescent intensity transected/fluorescent intensity control]*100). This value was calculated for three sections per marker per animal. The average and SEM for each marker across all sections was calculated, and P-values were calculated using a two-tailed student’s t-test.
Cell size calculations
ImageJ software was used to calculate size of DRG neurons. All images were taken at 20x magnification. To avoid bias in counting, the first 25 positive cells in each image from left to right were counted. In total, 225 cells per marker were counted (n = 3 animals, 9 DRG per animal). Using ImageJ, cells were outlined using the lasso tool and the area was obtained. For graphical representation, cells were divided into 100um2 bins.
Expression profile quantification
Expression profile of Nmb+ DRG neurons was evaluated by counting the number of cells that were positive for Nmb, co-staining markers, or both in 3 wild-type P14-P21 animals (6–8 lumbar DRG sections per animal). Total number of positive cells in each category was calculated for each animal, and the percentages of double positive neurons with regard to total Nmb+ neurons (Nmb+; costain+ /total Nmb+) or to total co-staining-marker-positive neurons (Nmb+; costain+ positive/total costain+) were calculated. The average and SEM of these percentages between animals were then calculated.