ondary antibodies ). 2.8. Statistical analysis were determined from ImageJ densitometry analysis and compared using Mann Whitney U tests. All F test statistics are described as a column factor with reference to drug/experimental grouping. NS designates not significant. 3. Results All data are represented as means SEM. Data were extracted and analyzed using Microsoft Excel 2010, Graphpad Prism v6 and ImageJ. Nociceptive behavioral analyses were between-subjects designs comparing effects of drugs by two way ANOVA with post-hoc Bonferroni tests. In those experiments involving intrathecal and intraperitoneal administration of drugs in nave animals, both hind paws were included in the analysis as replicates. EMG experiments used a within-subjects design and immunofluorescence experiments a between-subjects design with the effects of drug treatment compared to baseline values using one-way ANOVA 
with post-hoc Bonferroni tests. Immunofluorescence analysis of spinal cord was taken from entirety of dorsal horn. DRG and spinal cord neuron counts were ascertained from multiple representative images, at least 10 per animal and the mean value of those 10 calculated. Coloc2 analysis was used to ascertain the pixel intensity spatial correlation of SRSF1 and vGLUT1 staining in the spinal cord. This provides an automated measure of the correlation of pixel intensity for the two independent immunofluorescence channels for each sample, given as the Pearson’s correlation co-efficient. Western blot analyses of SRSF1 and VEGF-A family expression 3.1. SRSF1 is predominantly expressed in myelinated neurons in rats SRPK1 and SRSF1 are key factors in the control of VEGF-Axxxa preferential splicing particularly PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19840854 in disease. SRSF1 is expressed in the cytoplasm of dorsal root ganglia neurons in nave animals. Upon activation, SRSF1 is known to translocate from the cytoplasm to the GW 501516 nucleus, where it is involved in pre-mRNA processing. Following PSNI, SRSF1 immunoreactivity in sensory DRG neurons was found to be nuclear in some but not all neurons. Matched IgG and omission of primary antibody controls showed no signal. PSNI injury induces activating transcription factor 3 expression in injured DRG sensory neurons. There was an increase in ATF3-positive DRG neurons after PSNI, with 43% of DRG neurons expressing ATF3 post-PSNI compared to only 1% in nave animals. After PSNI, all nuclear localized SRSF1-positive DRG neurons were also ATF3 positive, indicating nuclear SRSF1 was exclusively found in damaged neurons. This represents that 45% of ATF3 -positive neurons were also SRSF1 Naive DAPI PSNI SRSF1 SRSF1 DAPI IgG Control DAPI DAPI Naive SRSF1 PSNI SRSF1 DAPI PSNI PSNI No Primary DAPI NeuN ATF3 NeuN ATF3 SRSF1 PSNI NeuN PSNI ATF3 SRSF1 ATF3 NeuN Fig. 1. SRSF1 expression and activation in DRG sensory neurons following PSNI injury. SRSF1 was expressed in the cytoplasm of the DRG sensory neurons in nave animals. Replacement of the primary antibody with a species matched IgG control DRG image resulted in no staining. SRSF1 was co-localized with nuclear stain DAPI in DRG sensory neurons following PSNI injury. In some neurons cytoplasmic SRSF1 is still evident. Omission of the primary antibody resulted in no staining. Representative examples of ATF3 expression in NeuN-co-labeled DRG sensory neurons in nave and PSNI animals. The number of ATF3 positive DRG neurons was significantly increased in the L4 from PSNI animals. High magnification representative images of SRSF1/ATF3/Ne