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The first objective was to determine correlation among various experimental and clinical pain measurement procedures. The second objective was to evaluate the influence of rotary instrumentation with continuous irrigation on pain and neuropeptide release levels.Forty patients who had preoperative pain at the levels of 3–8 on the visual analogue scale were included. Gingival crevicular fluid (GCF) samples were collected. Patients were randomly assigned to 2 treatment groups, the standard preparation group and the preparation with continuous irrigation group. Apical fluid samples (AFS) were collected after instrumentation. In the second visit, the patients' pain levels were recorded, and GCF and AFS were obtained. Substance P, calcitonin-gene related peptide (CGRP), interleukin (IL)-1β, and IL-10 levels were analyzed from the GCF and AFS samples. For comparison between groups, the Mann-Whitney test was used (P < .05).In terms of clinical data, no significant difference was detected in the first and second sessions between groups. The IL-10 level obtained from AFS significantly decreased in the second session in both groups (P < .001). Visual analogue scale scores of spontaneous pain correlated with percussion pain positively (r = 0.718, P < .001). CGRP (GCF) (second session) and IL-10 (GCF) (second session) positively correlated with percussion pain (r = 0.425, P < .01) (r = 0.379, P < .05).Rotary preparation with continuous irrigation has not been more effective than the standard preparation method for reducing pain. Because of determination of the correlation between CGRP and IL-10 with percussion pain, these neuropeptides can be used in further studies.