2.1 Experimental animals
Fifteen nine-month-old male Fisher 344/Brown Norway Cross inbred rats were used in this study. The rats had a mean body weight of 436 g (SD
= 45 g). Twelve rats underwent laryngoscopy and BoNT injection. Euthanasia and muscle harvest were performed at four time points (3 rats per time point): 72 hr, 7 days, 14 days, and 56 days post-injection. The initial and final time points were selected based on previous work demonstrating a robust chemodenervation effect by 72 hr post injection, with complete return of function by 56 days post injection [14
]. Three rats, employed as controls, underwent laryngoscopy and immediate euthanasia followed by muscle harvest.
2.2 Laryngeal visualization and BoNT injection
Anesthesia was induced using 2–3% isoflurane gas delivered at 0.8–1.5 L/min and maintained using an intraperiotoneal injection of ketamine hydrochloride (70 mg/kg) and xylazine hydrochloride (5 mg/kg). Atropine sulfate (52 µg/kg) was administered in order to reduce salivary secretions and assist with laryngeal visualization.
Laryngeal visualization was performed using a custom laryngoscope [20
] and operating platform [16
] in addition to a 1.9 mm diameter 25° rigid endoscope (Richard Wolf, Vernon Hills, IL) coupled to a halogen light source, display monitor and digital video camera (Sony DCR-PC100, Tokyo, Japan). Vocal fold opening and closure during respiration were recorded immediately prior to BoNT injection, at 72 hr post-injection and immediately prior to euthanasia and muscle harvest. These data were also collected from the control animals. A previous report documented that vocal fold movement during respiration is unaffected by the anesthesia cocktail used here [20
Commercial BoNT serotype A (BoNT/A) (Botox®, Allergan Pharmaceuticals, Irvine, CA) was reconstituted in 0.9% saline and injected at a concentration of 0.01 U in 0.4 µL, using a 5 µL syringe with a 50 mm, 26-gauge needle. Injections were unilateral, and made into the TA muscle under endoscopic guidance ().
Figure 1 Schematics and photographs illustrating (A) injection of BoNT/A into the left TA muscle using a 50 mm, 26-gauge needle and endoscopic guidance, and (B) measurement of maximum vocal fold opening (inspiratory) and closure (expiratory) angles during respiration. (more ...)
Measurements of vocal fold movement during respiration were employed to confirm BoNT effect, as illustrated in . All measurements were made using Photoshop 7.0 (Adobe Systems, San Jose, CA). Still frames were selected representing maximum vocal fold opening (during inhalation) and closure (during exhalation). A reference line was constructed between points at the anterior (A) and posterior (P) commissures. Two additional lines were drawn between P and points on the vocal fold medial edge at the right (R) and left (L) vocal processes. Angles APL and APR were measured and compared across the maximum opening and closure frames. Vocal fold movement angle was calculated by subtracting the maximum vocal fold closure angle from the maximum vocal fold opening angle, on a given side. Sample identity was masked and all measurements were performed in random sequence to control for any order effect. Inter- and intra-measurer agreement data were collected for 50% of all measurements.
2.3 Laryngeal harvest and dissection procedures
Euthanasia was performed via intracardiac injection of Beuthanasia (0.22 mL/kg). The larynx was harvested en bloc, and dissected using a surgical microscope and microsurgical instruments. The larynx was separated along the midline, allowing separate treatment of the left and right sides. A longitudinal incision was made in the subglottis, and the mucosa was undermined as far as the supraglottis to expose the TA muscle. The entire muscle body (muscularis and vocalis) was then harvested by dissection, proceeding anteriorly from the lateral base of the arytenoid cartilage to the thyroid cartilage. Following each dissection, the remaining hemilarynx was inspected to ensure that the non-TA intrinsic laryngeal muscles remained intact and undisturbed. The time duration from euthanasia to completion of dissection was approximately 10 min in all cases.
2.4 Sample preparation
Muscle samples were placed in 25 µL osmotic lysis buffer (0.3% SDS, 10 mM Tris; pH 7.4) containing 10% nuclease inhibitor (500 µg/mL RNase, 1 mg/mL DNase, 50 mM MgCl2, 100 mM Tris; pH 7.0) and 1% protease inhibitor (20 mM AEBSF, 1 mg/mL leupeptin, 360 µg/mL E-64, 500 mM EDTA, 560 µg/mL benzamidine) solutions. Tissue homogenization was performed on ice using an ultrasonic homogenizer (BioLogics Model 300V/T, Manassas, VA) for 6 min at 40% power with a micro tip. After the addition of 25 µL boiling buffer (5% SDS, 10% glycerol, 60 mM Tris; pH 6.8), the samples were placed in a boiling water bath for 30 min to facilitate dissolution, cooled on ice, and then centrifuged to pellet solids. The samples were then frozen and stored at −80° C in preparation for total protein quantitation and electrophoresis.
Total protein quantitation was performed spectrophotometrically using the bicinchoninic acid method [22
] and kit produced by Pierce Biotechnologies (Rockford, IL). BSA was employed as a standard and absorbance at 562 nm was measured using the Smart Spec 3000 spectrophotometer (BioRad Laboratories, Hercules, CA). Each assay was replicated, and final results were averaged. Mean final measurements of total protein ranged from 50–350 µg across all samples.
2D SDS-PAGE was performed using the carrier ampholine isoelectric focusing method [23
]. Total protein sample load was 50 µg for gels intended for silver staining and image analysis (one per animal, three per experimental time point), and 225 µg for a series of replicate gels (one per experimental time point) intended for Coomassie blue staining and spot excision. Isoelectric focusing was performed in glass tubes (2.0 mm inner diameter) using 2.0% pH 3.5–10 ampholines (Amersham Pharmacia Biotech, Piscataway, NJ) for 9600 Vh.
After equilibrium for 10 min in buffer (10% glycerol, 50 mM dithiothreitol, 2.3% SDS and 0.0625 M Tris; pH 6.8), each tube gel was sealed to the top of a stacking gel above a 7.5 mm thick 10% acrylamide slab gel. SDS slab gel electrophoresis was performed for approximately 4 hrs using 12.5 mA/gel. Six proteins (Sigma, St. Louis, MO) were added to the agarose used to seal the tube gel to the slab gel, as MW standards: Myosin (220 kDa), phosphorylase A (94 kDa), catalase (60 kDa), actin (43 kDa), carbonic anhydrase (29 kDa) and lysozyme (14 kDa). These standards appear along the basic edge of each gel. The gels were either silver [24
] or Coomassie blue stained. Gels were dried between cellophane paper.
2.6 Gel image analyses
Gels were scanned using an Agfa Arcus II flatbed scanner (Agfa, Mortsel, Belgium) in transparent mode, at 200 dpi resolution and 24-bit image depth. Image analysis was performed using Melanie 4.02 (GeneBio, Geneva, Switzerland) [25
]; and a previously reported analysis protocol [28
]. Briefly, automatic spot detection was performed and then refined by adjustment of the primary detection parameters (number of smoothing passes, saliency of the spot feature, minimum pixel area) in conjunction with visual inspection. Final parameter values, judged as providing optimal detection across all 15 gels, were 2 smoothing passes, a 3.5 saliency value, and 18 pixels minimum area. Following automatic detection, matching was performed for all silver-stained gels within their respective experimental groups (control, 72 hr, 7 days, 14 days, 56 days post-injection) and across the entire population of 15 silver-stained gels. The gel with the greatest number of protein spots in each experimental group was selected as the reference gel. Six widely distributed and visually salient proteins were selected as starting landmarks for the matching algorithm.
Spot-by-spot visual inspection and manual correction of detection and matching errors were performed by an expert gel analyst using each gel image, its 3-dimensional representation, and an intensity variation profile based on its entire 24-bit image depth. Sample identity was masked. All ratings were performed in random sequence to control for any order effect.
Synthetic master gels were constructed representing each of the 5 experimental groups. The gel with the greatest number of protein spots in each experimental group was again selected as the reference gel. Spots added to each synthetic gel were exclusive to those consistently detected and matched across all 3 constituent gels. Automatic spot matching, visual inspection, and manual correction were performed using the synthetic control gel as the reference.
Number of spots detected in each gel, and number and percentage of spots matched within the 5 experimental groups, 5 synthetic gels, and across all 15 gels were measured. Normalized spot volume was calculated using standard software algorithms. Estimated MW values for identified spots were determined by logarithmic interpolation and extrapolation of values for the MW standards present along the basic edge of each gel. Estimated pI values were determined by linear interpolation and extrapolation of surface pH measurements taken from four blank isoelectric focusing tube gels.
Qualitative observations of consistent protein spot presence/absence across experimental groups were noted during the visual inspection and manual correction of spot detection and matching. The purpose of this additional visual-perceptual analysis was to detect any potentially interesting spots that, due to their consistent absence in at least one experimental condition, were not detected by the spot matching algorithm and therefore not included in the quantitative analysis. Criteria for spot observations of potential importance were defined as follows: a) present in all 3 control gels and absent in all 3 gels in at least one other experimental group; or b) absent from all 3 control gels and present in all 3 gels in at least one other experimental group.
2.7 Protein spot identification
Spots of interest were manually excised from the Coomassie blue stained gels, transferred to clean tubes and rehydrated in Milli-Q water (Millipore, Bedford, MA). Spots were prepared for digestion by washing twice with 100 µL 0.05 M Tris, pH 8.5/30% acetonitrile for 20 min with shaking, then with 100% acetonitrile for 1–2 min, before drying for 30 min in a Speed-Vac concentrator.
Samples were digested using 0.06 µg modified trypsin (sequencing grade, Roche Molecular Biochemicals, Indianapolis, IN) in 13–15 µL 0.025 M Tris, pH 8.5 and then incubated at 32° C overnight. Peptides were extracted using 2× 50 µL 50% acetonitrile/2% TFA and the combined extracts were dried and resuspended in 3 µL matrix solution consisting of 10 mg/mL 4-hydroxy-α-cyanocinnamic acid in 50% acetonitrile/0.1% TFA and containing two internal standards, angiotensin and ACTH 7–38 peptide. Following resuspension, 0.5 µL of each sample was spotted onto a MALDI plate and, once dry, washed twice with Milli-Q water. MALDI-MS was performed on the digested samples using a Voyager DE Pro mass spectrometer (Applied Biosystems, Foster City, CA) in the linear mode.
Peptide mass data were analyzed using ProFound (http://prowl.rockefeller.edu
) and the MS-Fit module of the ProteinProspector program (http://prospector.ucsf.edu
, version 4.0.8), and the NCBI and/or GenPept protein databases. Mass range was set at 900–5000 Da and mass error tolerance was set at 0.5 Da. Minimum fragment length was 5 peptides. A maximum of one missed cleavage was allowed. Acrylamide modified cysteine residues were allowed.
2.8 Statistical analyses
Vocal fold movement angle data were analyzed using Wilcoxon rank sum tests. Inter- and intra-measurer agreement data were analyzed using the Bland-Altman procedure with limits of agreement set at 95% [29
]. Percentage of spots matched across gels was calculated using a previously reported algorithm [27
]. Statistical comparisons were performed for each group of matched spots in the standard (i.e., non-synthetic) gels using a series of one-way ANOVAs, with experimental group as a fixed effect. Post-hoc pairwise comparisons of significant main effects were then performed for each post-injection experimental group against the control group. A parallel analysis using the synthetic gel set was conducted by calculating percentage change in normalized spot volume for each post-injection gel compared with the control gel. An α-level of 0.01 was employed for all statistical testing.