Ovarian Hormone Modulation of Itch Sensitivity

All experimental procedures were approved in accordance with the Guide for the Care and Use of Laboratory Animals (44 ) prepared by Okayama University (Okayama, Japan), by Kyoto Prefectural University of Medicine (Kyoto, Japan), by Toyama University (Toyama, Japan), and by the National Institute of Genetics (Shizuoka, Japan) and performed in accordance with the NIH guidelines on animal care. Adult wild-type Wistar rats and GRPR-mRFP transgenic rats were used in this study. To examine the effects of female sex steroid hormones, ovariectomized females were implanted with a blank capsule, a capsule containing physiological levels of 17β-estradiol, a capsule containing physiological levels of progesterone, or a capsule containing physiological levels of both hormones, for 1 to 2 mo. For itch behavioral analysis, rats received either saline, 3% histamine, or 10% CQ diphosphate salt diluted in saline via intradermal injection in the nape of the neck, cheek, or hind paw. Immediately after the injection, the rat was placed into the arena and videotaped from above for 60 min for scratching behavior. Mechanical sensitivity was assessed by the von Frey filament test, and thermal pain sensitivity was assessed by the Hargreaves test. For RT-PCR, enzyme-linked immunosorbent assay, Western blot, and the ChIP assay, the dorsal horns of the cervical spinal cords were collected and used for analysis. Complete methods are described in SI Appendix. Brain and spinal cord sections were used for immunofluorescence and immunoperoxidase histochemistry after perfusion with physiological saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer. Antibody information is provided in SI Appendix, Fig. S2. In vivo extracellular single-unit recordings of superficial spinal dorsal horn (lamina I and II) neurons were performed in female rats. Statistical analyses were performed using SPSS Statistics version 27 (IBM). Graphs were made using GraphPad Prism 8 (GraphPad Software). Statistical analyses for each study are indicated in the figure legends. More detailed information on materials and methods is provided in SI Appendix.