Injection of formalin in the rat hindpaw produces two phases of nociceptive behavior. Although it is generally agreed that the first phase results from direct chemical activation of nociceptive primary afferent fibers, the factors that contribute to the second phase are not established. In the present study, we monitored the expression of the c-fos protein to evaluate whether the pattern of activity of dorsal horn neurons differs as a result of ongoing afferent activity during the two phases. To selectively block the first or second phase, we respectively used remifentanil, a potent and short acting opiate agonist, and QX-314, a quaternary derivative of lidocaine, which does not cross the blood brain barrier. We also evaluated the effect of eliminating nociceptive behavior in both phases using both remifentanil and lidocaine or a combination of local anesthetics, bupivicaine and quaternary lidocaine. In all groups, formalin (5%, 50 microliters) was injected subcutaneously into the plantar surface of the hindpaw. To assess the nociceptive behavior produced by formalin, we monitored the number of flinches. Injection of remifentanil during the first phase completely blocked the first phase formalin-evoked nociceptive behavior, and had no effect on the second phase. Injection of lidocaine during the interphase completely blocked second phase nociceptive behavior. As expected, when remifentanil was administered during the first phase and lidocaine during the second phase, all formalin-evoked nociceptive behavior was blocked. The same was true for rats that received injections of bupivicaine and lidocaine during phases 1 and 2, respectively. In laminae I-II of the L4-L5 segment, the magnitude of the decrease in Fos expression was comparable for remifentanil (26.5%) and lidocaine (27.3%); the decrease was greater when both remifentanil and lidocaine were administered (50.5%), and even greater when bupivicaine and lidocaine were used (74.2%). In laminae V-VI, remifentanil, by itself, decreased c-fos expression by 39.4%; for lidocaine alone, the decrease was 58.4%. We did not observe further significant decreases when both remifentanil and lidocaine, or bupivacaine and lidocaine were injected (69.7% and 74.6%, respectively). Our results not only provide strong evidence that activity during the second phase is necessary for maintaining the maximal expression of c-fos in the spinal cord, but also reveal significant regional differences in the central patterns of activity generated during the two phases. These results also confirm our previous reports that c-fos expression is not eliminated when the behavioral manifestation of the noxious stimulus is completely blocked.

Hindpaw injection of formalin produces acute (Phase 1) and persistent (Phase 2) nociceptive behaviors. This model has provided critical evidence supporting a contribution of central sensitization (hyperexcitability of spinal neurons) to the expression of persistent pain. Here, we evaluated the contribution of ongoing peripheral nerve inputs to Phase 2 pain responses. In addition to pain behavior (flinching), we measured formalin-evoked increases in arterial pressure and heart rate; these cardiovascular responses were also biphasic in nature. The arterial pressure response correlated highly with behavior, and was dependent on formalin concentration (0.625–5.0%), indicating that it was largely driven by noxious input. Lightly anesthetized (0.7% halothane) rats exhibited robust increases in blood pressure in the absence of pain behavior, indicating cardiovascular responses did not reflect somatomotor-cardiovascular coupling. Animals obtained from Charles River exhibited slightly larger Phase 2 flinching and heart rate responses compared to those obtained from Bantin and Kingman, suggesting cardiovascular-related pain responses can vary with the source of animal. We next evaluated the contribution of ongoing peripheral nerve activity to the expression of the Phase 2 pressor, tachycardia, and flinch responses. After Phase 1 subsided, but before Phase 2 began, we locally anesthetized the ipsilateral or contralateral (control) hindpaw with a hydrophilic lidocaine derivative, QX-314 (2%). Intraplantar QX-314 blocked Phase 2 pressor, tachycardia and behavioral responses only when injected into the paw that received formalin (2.5% or 10.0%). We conclude that persistent ongoing activity in peripheral afferent fibers during Phase 2 is required for the persistent pain evoked by formalin.

The opioid antagonist, naloxone, produces equivocal effects on the magnitude of nociceptive responses in several animal models of persistent pain, including the formalin test. Hindpaw injection of dilute formalin produces not only inflammation but also phasic (Phase 1) and persistent (Phase 2) behavioral and cardiovascular nociceptive responses in the rat. To test the hypothesis that endogenous opioid systems contribute to the magnitude of responses to intraplantar formalin injection, we evaluated the effects of continuous naloxone administration (0.01-100 mg/kg per h, i.v.) on formalin-evoked hindpaw inflammation, on behavioral indices of pain, flinching and licking pain behavior, and on changes in mean arterial pressure and heart rate. We report that naloxone, at doses less than 100 mg/kg per h, did not change any formalin-evoked response. Although the 100 mg/kg per h dose significantly decreased these responses, it also produced muscle rigidity and profound bradycardia. We conclude that endogenous opioids do not significantly modulate the nociceptive processing induced by subcutaneous formalin.

Background: MetaSite Breast™ is a validated assay to predict risk of distant breast cancer metastasis in patients with HR+/HER2- ESBC. The assay measures the number of MetaSites defined as tumor microanatomic structures composed of MENA protein expressing tumor cells in contact with CD31+ endothelial cells and CD68+ macrophages. Previous studies have demonstrated that an increased number of these microanatomic structures is associated with distant metastasis (DM) in HR+/HER2- ESBC independent of clinicopathologic features. Analytical validation of MetaSite Breast™ demonstrated precision of 97-99% (repeat image analysis of the same slide) and performance of 91-96% (staining and image analysis of serial tumor sections). We sought to further understand the importance of the MetaSite in predicting distant breast cancer metastasis utilizing a fully automated prognostic assay in an independent large patient cohort. Methods: We conducted a nested case-control study within a cohort of 3,760 patients diagnosed between 1980 and 2000 with invasive breast cancer from the Kaiser Permanente Northwest health care system. Cases (n=259) were women who developed a subsequent distant metastasis; controls, selected using incidence density sampling, were matched closely to cases (1:1) on age at and calendar year of primary diagnosis. Of the 481 patient tumor samples evaluated in this study, 57% were HR+/HER2-, 19% were triple negative (TN), and 15% were HER2+ disease. Multivariate models were adjusted for clinical factors including: lymph node status, tumor size, tumor grade, and HRT; as well as matching variables: age and year of diagnosis. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated using logistic regression. Results: In the HR+/HER2- group, MetaSite Score (MS) ranged from 0-357 and the mean was 44.6. MS was a significant predictor of DM (P=0.039) in patients with HR+/HER2- disease. Cut-points based on tertiles of MS in all 259 controls defined intermediate (13-41) and high (>41) risk groups that were significantly associated with risk of DM versus the low risk group (OR=2.24; 95%CI=1.23-4.13, P=0.009) and (OR=2.94; 95%CI=1.62-5.41, P=0.0005), respectively. Univariate estimates of absolute risk of DM with cutoffs based on 90% sensitivity and specificity were 9.4% for the low risk group (MS<7), 14.1% for the intermediate (MS=7-91), and 23.4% for the high (MS>91). When adjusted for clinical factors, estimates of absolute risk of DM were 6.6%, 14.1%, and 33.0% for the low, intermediate, and high risk groups, respectively. A binary cut-point for the high risk group was determined (MS>14) and was significant with a 2-fold higher risk of DM versus the low risk group and adjusted for clinical covariates (P=0.036). MS was not positively associated with DM in TN or HER2+ disease. Conclusions: MetaSite Breast™ significantly predicted the risk of distant breast cancer metastasis in ESBC patients with HR+/HER2-disease, independent of classical clinicopathologic features. Citation Format: Donovan MJ, Jones JG, Entenberg DR, Condeelis JS, D'alfonso TM, Gustavson M, Molinaro A, Oktay MH, Xue X, Sparano JA, Peterson MA, Podznyakova O, Rohan TE, Shuber AP, Gertler FB, Ly A, Divelbiss ME, Hamilton DA. Analytical and clinical validation of a fully automated tissue-based quantitative assay (MetaSite Breast™) to detect the likelihood of distant metastasis in hormone receptor (HR)-positive, HER2-negative early stage breast cancer (ESBC) [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-05-06.