jured nerve, 8 rats underwent PSNI surgery and were treated with the SR protein kinase inhibitor, SRPIN340 or vehicle. SRPIN340 was incorporated into a sterile gel consisting of, 0.0006% benzalkonium chloride) and applied to the nerve in the area of the tight ligation; gel without SRPIN340 was used in control animals. Nociceptive testing was performed on days 1 and 2 after PSNI. Animals were killed by anesthetic overdose and the saphenous nerves and L3/L4 DRGs removed. VEGF-A splice variant mRNA expression was determined at the site of injury by qRT-PCR for total VEGFA and VEGF-A165a. D) Interactions between VEGF-A and TRPV1: i) Effect of pharmacological TRPV1 receptor blockade: Mechanical nociceptive behavior was determined every other day for 5 days. VEGF-A and SB366791 or vehicle were given via systemic injection on days 1 and 3 immediately after behavioral testing. To exclude an effect of pharmacological inhibition on central rather than peripheral TRPV1, 2.5 nM VEGF-A165a was injected subcutaneously into the plantar surface of the hindpaw was with 
either PBS or 1 M SB366791 followed by behavioral testing. ii) VEGF-A-mediated TRPV1 sensitization: To determine the effect of VEGF-A isoforms administered together with a non-sensitizing concentration of TRPV1 agonist in identified primary afferents, a bolus of 10 M capsaicin was injected through an intra-femoral arterial cannula inserted in the mid-thigh in the opposite hind limb to the recordings, with the tip advanced to the bifurcation of the descending aorta. This allowed close arterial delivery of capsaicin to the peripheral afferent receptors. The effects of VEGF-A165a and VEGF-A165b on TRPV1 agonist responses were investigated using close arterial injection of capsaicin combined with local injection of 2.5 nM VEGF-A165a, 2.5 nM VEGF-A165b or both together. When VEGF-A165b was used, it was then followed by VEGF-A165a to confirm VEGF receptor responses in the afferents studied, as described above. Numbers of afferents included in the experiments were: ongoing and mechanically evoked activity — saline vehicle n = 12, VEGF-A165a n = 7, and VEGF-A165b n = 5; mechanical activation threshold — VEGF-A165a, saline n = 7, VEGF-A165b n = 5; capsaicin sensitization — baseline n = 16, VEGF-A165a n = 8, VEGF-A165b n = 8, VEGF-A165a + VEGFA165b n = 7. Experimental note: This concentration of capsaicin delivered by close arterial injection does not result in sensitization or desensitization of the TRPV1 receptors to agonist stimulation on repeated injection. A low capsaicin concentration to avoid possible desensitization of TRPV1 in the presence of a further sensitizing agent, as we hypothesized VEGF-A165a to be. Capsaicin injection resulted in a short burst of action potentials that confirmed access of the agonist to the afferent receptor terminals. It should be noted that the effective concentration of capsaicin at the primary afferent KU55933 web terminals when delivered by this method is approximately 1000 fold lower than that injected as a result of dilution in hind limb blood volume, and tissue penetration. Statistical analyses The majority of data sets was Gaussian in nature and therefore met the requirements for parametric analyses; in a small number of cases, data sets were log transformed to render them Gaussian prior to analysis. Multiple groups were compared using one or two way ANOVA followed by post-hoc Bonferroni tests where PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19839935 appropriate, and where Gaussian assumptions were not met or log t
