Role of nitric oxide and Jun N-terminal kinase in the development of dark neurons in the dorsal horn of the spinal cord following induction of inflammatory pain

Introduction: Dark neurons which their morphological characteristics are consistent with those of cells undergoing apoptosis, are generated as an acute or delayed consequence of several pathological situations. The present study was designed to evaluate whether inflammatory pain regarding the role of NO and JNK lead to the formation of dark neurons in the dorsal horn of the lumbar spinal cord in rats. Methods: Acute or chronic inflammatory pain was induced by intraplantar injection of 1%, 2.5% or 5% formalin in male Wistar rats weighing 250-300 g (n=7). Spectrophotometrical analysis of the serum nitrite (metabolite of NO) and histological procedures for detection of dark neurons were performed at definite time intervals. Pretreatment with celecoxib 10, 20 or 40 mg/kg/i.p.; quercetin 40 or 100 μg/rat/i.t. as an inhibitor of JNK pathway, and PTIO 20 or 30 μg/rat/i.t, as NO scavenger, were performed to investigate the role of NO and JNK. Results: Injection of formalin led to the increase of the serum nitrite in the concentration and time-dependent manners. The effect of 5% formalin was significantly eminent which was blocked by celecoxib 40 mg/kg. Visual inspections of the spinal cord sections showed that on day 5, following chronic injections of 5% formalin, numbers of dark neurons were significantly increased in the lumbar dorsal horn. Acute and chronic administration of other concentrations of formalin did not induce any remarkable dark phenotype. Injections of celecoxib 40 mg/kg, quercetin 100 μg/rat/i.t. or PTIO 30 μg/rat/i.t. before each injection of 5% formalin, led to a reliable reduction of dark neurons. Conclusion: The results showed that the intensity and duration of the inflammatory pain play a major role in its peripheral and central developed disorders. According to the role of NO and JNK; it seems that administration of their inhibitors, or an appropriate dose of celecoxib may exert a protective effect against the aforementioned consequences.