Computational protocol: Vagus nerve stimulation inhibits trigeminal nociception in a rodent model of episodic migraine

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Protocol publication

[…] Immunostaining procedures and analysis were performed essentially as described in previous studies.,, Slides containing 1 tissue section from each experimental condition were covered with 1 × phosphate-buffered saline for 5 minutes before incubation for 20 minutes in 5% normal donkey serum (Jackson ImmunoResearch Laboratories, West Grove, PA) in phosphate-buffered saline containing 0.1% Triton. Primary antibodies diluted in 5% normal donkey serum and Alexa Fluor secondary antibodies (Invitrogen; 1:200 dilution in PBS, Carlsbad, CA) were incubated with tissues as reported in Table . Tissues were mounted in the VECTASHIELD medium containing 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA). A Zeiss AxioCam MRm camera (Carl Zeiss, Thornwood, NY) mounted on a Zeiss Imager Z1 fluorescence microscope was used to collect a single 4 × 3 tiled ×200 image of the dorsal medullary horn region and 3 × 3 tiled ×200 images of the V1/V2 area of the TG. Zen 2011 software (Carl Zeiss) was used to evenly balance the background of each image before densitometric analysis of gray scale JPEG images using ImageJ software. For spinal cord tissues, integrated densities were acquired from 3 independent experiments by measuring pixel densities in 10 nonoverlapping, rectangular regions of interest encompassing laminas I to III for each image. In addition, background measurements, which were acquired from acellular areas as determined by DAPI staining, were averaged and subtracted from the regions of interest values. Relative average mean values were determined for each condition and data were reported as average fold change ± SEM relative to the average mean for control animals, which was set equal to 1. Similar methods were used to quantitate P-ERK expression in primary trigeminal nociceptors using background measurements acquired from areas containing only Schwann cells and neural fibers in the ganglion as determined by DAPI staining. For quantification of nuclear P-ERK in the TG, the number of neuronal cells exhibiting nuclear localization of P-ERK was divided by the total number of visible neuronal nuclei as identified by DAPI staining. Results are reported as the average percent ± SEM of neurons with P-ERK nuclear staining. [...] For nociception experiments, we anticipated a medium effect size (η2 = 0.083). This study is a factorial design of 3 (Groups) × 4 (Time points). These groups included a set of animals without any additional experimental conditions (naive), a group involving trigeminal sensitization induced by neck muscle inflammation and later activated by an odor trigger (muscle + CBL) and a group that received both forms of stimulation and receiving transdermal VNS (muscle + CBL + VNS). Using G-Power (Heinrich-Heine-University, Düsseldorf, Germany) to conduct a power analysis and using a power of 0.80 at α = 0.05, the minimum sample size to detect expected main effect and interactions required at least 24 animals (8 naive, 8 M+CBL, 8 M+CBL+VNS). Outliers were determined by SPSS Statistics 21 software (IBM, North Castle, NY) using 3 as a multiplier. To determine the normality of behavioral data sets, a Shapiro-Wilk test was used, whereas a Levene test was used to determine equal or unequal variance. Because it was determined that data sets were in violation of these assumptions, a nonparametric statistical analysis was used to determine significant changes. To determine if the observed effects were statistically significant, a Kruskal-Wallis analysis of variance (ANOVA) was performed followed by a Mann-Whitney U post hoc test with a Bonferroni correction (αaltered = 0.05/3, P < 0.017) for pairwise comparisons at each time point. In addition, a Friedman ANOVA was performed within each group, followed by a Wilcoxon signed-rank test with a Bonferroni correction (αaltered = 0.05/3, P < 0.017) to evaluate comparisons made in a single group from baseline readings. All statistical tests were conducted using SPSS Statistics software.For the immunohistochemical studies, we anticipated a large effect size (η2 = 0.702) based on previously published data. Using a power of 0.80 at α = 0.05 alpha, the minimum sample size needed to detect the expected main effect would require a minimum of 12 animals (4 naive, 4 M+CBL, 4 M+CBL+VNS). Any values detected as outliers by SPSS using 3 as a multiplier were removed from analysis. A Shapiro-Wilk test was used to determine data set normality, whereas a Levene test was used to determine equal or unequal variance. Data sets were found to adhere to the assumption of normality; however, not always to the assumption of equal variance. Therefore, statistical differences were determined using a 1-way ANOVA, supported by both Welch and Brown-Forsythe tests, with either a Tukey or a Games-Howell post hoc test in SPSS software, and considered different when P < 0.05. […]

Pipeline specifications

Software tools ImageJ, SPSS
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Rattus norvegicus, Homo sapiens
Diseases Migraine Disorders, Muscular Diseases, Peripheral Nervous System Diseases, Ganglion Cysts