Human psychophysics: subjects and pruritogens

32 healthy volunteers, 15 females and 17 males, were enrolled after providing written informed consent to protocols approved by the Yale University Human Investigation Committee and that met the Declaration of Helsinki Principles for human subjects. The mean age was 32.8 + 14.9, the median 28 years. Among these 19 were Caucasian, 8 Asian, 2 Hispanic, and 3 were more than one race. Initially, 33 subjects were recruited, 1 did not qualify for participation due to not feeling itch during tests with a single pruritogen and data from 3 subjects were not included in the analysis because all stimuli could not be tested due to scheduling issues.

Potential subjects were excluded from participation in the study if they reported a medical history of cardiac, immunological, dermatological, or neurological disorders or reported taking antihistamine or analgesic medication recently.

The pruritogens, ALA (Sigma-Aldrich; 90 µg/10 µl; 100 mM; 1.01 µmol), BAM8-22 (Tocris Bioscience; 1 µg/10 µl; 51 µM; 0.51 nmol), and HIS dihydrochloride (Sigma-Aldrich; 10 µg/10 µl; 5.4 mM; 54 nmol), as well as a combination of two substances (ALA 90 µg with BAM8-22 1 µg in 10 µl) and combination of all three substances (ALA 90 µg with BAM8-22 1 µg and HIS 10 µg in 10 µl) were each prepared under sterile conditions in saline 0.9%. Effects of vehicle solutions (saline, ECF) were not tested as in a previous study normal saline elicited a transient itch of low magnitude in a small proportion of subjects tested (Simone et al., 1987). Similarly, in a previous study (Sikand et al., 2011b) ECF-induced sensations were minimal with an average peak magnitude itch that did not differ from itch induced by 10⁻³ µg of HIS and nociceptive sensations less than those produced by HIS doses ≥ 10⁻² µg. In the present experiments, we did not measure the pH of each solution. But in a previous study no difference was found in the itch evoked by HIS in a vehicle of normal saline alone (with pH of 5.0) versus a vehicle of phosphate buffered saline that had a pH of 7.4 (Simone et al., 1987). The testing procedure was performed as previously described (LaMotte et al., 2009; Sikand et al., 2009), and concentrations for injections were based on previous studies (Liu et al., 2012; Sikand et al., 2011a; Simone et al., 1991). After disinfection with an ethanol wipe, subjects received a superficial intradermal injection of 10 µl pruritogen solution into the volar forearm with the needle inserted approximately parallel to the skin and the tip of the needle visible through the skin (Sikand et al., 2011b). Each arm was tested maximally up to two times per session with subsequent injections made well outside the area of dysesthesias produced by a prior injection and after the sensation of the previous injection was not present anymore. The order of administration for trials was random and blinded for each subject. Control peptides, amino acids, and vehicles (BAM8-18, L-alanine, and saline) were not administered as their evoked responses were either minimal or absent in previous human psychophysics studies (Liu et al., 2012; Sikand et al., 2011a; Simone et al., 1987).

Subjects were instructed not to scratch or move the arm following the injection. A visual barrier was placed following the injection to cover the view on the injected arm, removing biases in ratings of sensations by observed visual changes to the skin.