Effects of Intracerebroventricular and Intra-Arcuate Nucleus Injection of Ghrelin on Pain Behavioral Responses and Met-Enkephalin and β-Endorphin Concentrations in the Periaqueductal Gray Area in Rats

Abstract

Ghrelin is an endogenous ligand for orphan growth hormone secretagogue receptors. Ghrelin receptors have been found in central nervous system (CNS) areas responsible for pain modulation and transmission. This study investigated the effects of intracerebroventricular (ICV) and intra-arcuate nucleus (ARC) injection of ghrelin on pain behavioral responses and levels of β-endorphin (β-EP) and met-enkephalin (MENK) in the periaqueductal gray area (PAG) during the formalin test in rats. Thirty-five male rats were studied in five groups. Ghrelin was injected into the left lateral ventricle (ICV, 5 µL) or into the ARC (1 µL). After 15 min, formalin (2.5%) was subcutaneously injected into the left hind paw. Behavioral nociceptive scores were recorded for 60 min. MENK and β-EP were collected by microdialysis in the PAG and determined by high-performance liquid chromatography (HPLC). ICV and ARC injection of ghrelin significantly reduced pain in all phases of the formalin test (p < 0.001). Dialysate concentrations of MENK and β-EP in the PAG increased in all the phases (p < 0.01). In conclusion, the present study shows that the ARC nucleus and the endogenous opioid system are involved in ghrelin-induced pain modulation.

Keywords: beta-endorphin; formalin test; ghrelin; met-enkephalin; microdialysis.

Figure 1

Time courses of the pain scores recorded in the control groups (Saline-lateral ventricle (ICV), Saline-ventromedial arcuate nucleus (ARC), SHAM) during the formalin test (60 min) divided into 5-min periods. Data are mean ± standard error of the mean (SEM).

Figure 2

(A) Time courses of the pain scores recorded during the formalin test in animals treated with Saline-ICV or Ghrelin-ICV. (B) Time courses of the pain scores recorded during the formalin test in animals treated with Saline-ARC or Ghrelin-ARC. Data are mean ± SEM. ** p < 0.001 and * p < 0.05 vs. other group, same time period.

Figure 3

(A) Time courses of the β-endorphin (β-EP) dialysate levels determined before, during and after the formalin test in animals treated with Saline-ICV or Ghrelin-ICV. (B) Time courses of the β-EP dialysate levels determined before, during and after the formalin test in animals treated with Saline-ARC or Ghrelin-ARC. Data are mean ± SEM. ** p < 0.001 and * p < 0.05 vs. other group, same time period.

Figure 4

(A) Time courses of the met-enkephalin (MENK) dialysate levels determined before, during and after the formalin test in animals treated with Saline-ICV or Ghrelin-ICV. (B) Time courses of the MENK dialysate levels determined before, during and after the formalin test in animals treated with Saline-ARC or Ghrelin-ARC. Data are mean ± SEM. ** p < 0.001 and * p < 0.05 vs. other group, same time period.

Figure 5

Graphical representation of the neural pathways considered in the study.

Figure 6

Schematic representation of the experimental design. The microdialysis and formalin test were performed 24 h after implantation of the microdialysis probe. Sample 1 (S1, 30 μL) was collected as the baseline value, and then Saline/Ghrelin were injected into the lateral ventricle (ICV) or arcuate nucleus (ARC) (S2). Dialysate samples were collected every 15 min for 2 h, the last sample (S8) being 15 min after the end of the formalin test. The formalin test was started after the optimal time for the outcome of the drug (15 min), and pain scores were recorded every 5 min for one hour.