![An increase in the amount of DAGLβ in blood cells and the accumulation of 2-AG in plasma contributes to cold-evoked hyperalgesia in HbSS(nh) mice. Data for biochemical studies were collected 2 hours after exposure to cold. (A) In contrast to HbAA mice, the level of 2-AG increased in plasma of HbSS(nh) mice exposed to cold. Numbers inside bars indicate group size. ∗Different from other groups (F[3,12] = 8.564, P = .003, 1-way ANOVA with Bonferroni t test. (B) The accumulation of 2-AG in the plasma of HbSS(nh) mice was associated with an increased level of DAGLβ in blood cells. The relative level of DAGLβ protein was defined as the amount of immunoreactivity in the HbSS(nh) sample/average amount of immunoreactivity in the HbAA sample × 100%. ∗Different from other groups (F[3,12] = 7.401, P = .005, 1-way ANOVA with Bonferroni t test). (C) Representative images of immunoreactive bands corresponding to DAGLβ isolated from blood cells [top, HbSS(nh) mice (a,d) and HbSS+cold mice (b,c,e)] and the total protein stain for loading control (bottom). DAGLβ was detected with rabbit anti-DAGLβ (1:500, Abcam). The secondary antibody was IRDye 800CW goat anti-rabbit (1:15 000; LI-COR). (D) The basal level of COX-2 protein in blood cells was higher in HbSS(nh) compared with HbAA mice. ∗Different from HbAA and HbAA+cold groups (F[3,16] = 20.530, P < .001, 1-way ANOVA with Bonferroni t test). Exposure to cold did not cause additional changes in HbSS(nh) mice. There was no difference in the level of COX-2 between naïve HbAA mice and HbAA mice exposed to cold. COX-2 was detected with rabbit anti-COX-2 (1:500, ABclonal). The secondary antibody was IRDye 800CW goat anti-rabbit (1:15 000; LI-COR). Numbers inside bars indicate group size. (E) Unlike vehicle, systemic administration of KT109, an inhibitor of DAGLβ, prevented acute mechanical hyperalgesia in HbSS mice. KT109 (30 μg) or vehicle (dimethyl sulfoxide: Tween 80:Saline [30:1:69]) was administered by intraperitoneal injection 1 hour before exposure to cold. ∗Different from BL at P = .002 (F[4,36] = 5.187) and #different from vehicle at P = .007 (F[1,36] = 11 852), 2-way repeated-measures ANOVA with Bonferroni t test, n = 5 to 6 mice/group.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/140/16/10.1182_blood.2022017309/13/blood_bld-2022-017309-gr2.jpeg?Expires=1735514520&Signature=C4IT5gTeLbud0O7-~DTi-CDoVLPKdm6c8HBZ-3r~o~36hWkYrfzGHKrUbpVYqDpFAy2-LT44CYhmMPS9Fs6yq91kjzcVwklW6G0xRtbgCY0szJlXkDKo57~ww67P2TDGUx6B-of6PO54Flpow4xY83O2rc0BF8fdVViiKBgqgK~jnYgd49kM3k8agwwbg7JBC0vTYyafUiI14s3TjR96Kw7LrE8PGYI9qhn-kK5L-lHgWoTUYCrIMrxmfDZisnvY5qD6dAPEXitJupaaWUYRTTE~gjeVo6KiFFH1QfKIoQQWcZx-VLvALsTefk7~0tEkShzCFtBRcx1ZdO564DS1mw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
An increase in the amount of DAGLβ in blood cells and the accumulation of 2-AG in plasma contributes to cold-evoked hyperalgesia in HbSS(nh) mice. Data for biochemical studies were collected 2 hours after exposure to cold. (A) In contrast to HbAA mice, the level of 2-AG increased in plasma of HbSS(nh) mice exposed to cold. Numbers inside bars indicate group size. ∗Different from other groups (F[3,12] = 8.564, P = .003, 1-way ANOVA with Bonferroni t test. (B) The accumulation of 2-AG in the plasma of HbSS(nh) mice was associated with an increased level of DAGLβ in blood cells. The relative level of DAGLβ protein was defined as the amount of immunoreactivity in the HbSS(nh) sample/average amount of immunoreactivity in the HbAA sample × 100%. ∗Different from other groups (F[3,12] = 7.401, P = .005, 1-way ANOVA with Bonferroni t test). (C) Representative images of immunoreactive bands corresponding to DAGLβ isolated from blood cells [top, HbSS(nh) mice (a,d) and HbSS+cold mice (b,c,e)] and the total protein stain for loading control (bottom). DAGLβ was detected with rabbit anti-DAGLβ (1:500, Abcam). The secondary antibody was IRDye 800CW goat anti-rabbit (1:15 000; LI-COR). (D) The basal level of COX-2 protein in blood cells was higher in HbSS(nh) compared with HbAA mice. ∗Different from HbAA and HbAA+cold groups (F[3,16] = 20.530, P < .001, 1-way ANOVA with Bonferroni t test). Exposure to cold did not cause additional changes in HbSS(nh) mice. There was no difference in the level of COX-2 between naïve HbAA mice and HbAA mice exposed to cold. COX-2 was detected with rabbit anti-COX-2 (1:500, ABclonal). The secondary antibody was IRDye 800CW goat anti-rabbit (1:15 000; LI-COR). Numbers inside bars indicate group size. (E) Unlike vehicle, systemic administration of KT109, an inhibitor of DAGLβ, prevented acute mechanical hyperalgesia in HbSS mice. KT109 (30 μg) or vehicle (dimethyl sulfoxide: Tween 80:Saline [30:1:69]) was administered by intraperitoneal injection 1 hour before exposure to cold. ∗Different from BL at P = .002 (F[4,36] = 5.187) and #different from vehicle at P = .007 (F[1,36] = 11 852), 2-way repeated-measures ANOVA with Bonferroni t test, n = 5 to 6 mice/group.
