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. 2022 Oct 20;140(16):1826-1830.
doi: 10.1182/blood.2022017309.

A model of painful vaso-occlusive crisis in mice with sickle cell disease

Iryna I Khasabova  1 , Joseph Juliette  1 , Victoria M Rogness  1 , Sergey G Khasabov  1 , Mikhail Y Golovko  2 , Svetlana A Golovko  2 , Stacy Kiven  3 , Kalpna Gupta  3   4 , John D Belcher  4 , Gregory M Vercellotti  4 , Virginia S Seybold  5 , Donald A Simone  1
Affiliations

A model of painful vaso-occlusive crisis in mice with sickle cell disease

Iryna I Khasabova et al. Blood. .
No abstract available

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Conflict of interest statement

Conflict-of-interest disclosure: K.G. received honoraria from Tautona Group, Novartis, and CSL Behring; and research grants from Cyclerion, 1910 Genetics, Novartis, Grifols, UCI Foundation, and SCIRE Foundation. The remaining authors declare no competing financial interests.

Figures

Figure 1
Figure 1.
Exposure to cold induced painful VOC in nonhyperalgesic HbSS mice. (A) Mechanical hyperalgesia appeared in HbSS(nh), but not in HbAA mice, 1 hour after exposure to cold and persisted for at least 24 hours. ∗Different from BL at P = .002 (F[3,40] = 3916) and #different from HbAA mice at P = .002 (F[1,40] = 18.1), 2-way repeated-measures ANOVA with Bonferroni t test, n = 5 to 7 mice/group. (B) Unlike HbAA mice, HbSS(nh) mice exhibited heat hyperalgesia, which began 1 hour after exposure to cold and disappeared by 24 hours. ∗Different from BL at P = .004 (F[4,44] = 4.57) and #different from HbAA mice at P = .002 (F[1,44] = 15.5, 2-way repeated-measures ANOVA with Bonferroni t test, n = 6 to 7 mice/group). (C) Exposure to cold caused prolonged deep tissue hyperalgesia in both sexes of HbSS(nh) mice. Because means of grip force were lower in female mice compared with male mice in both groups (†different from female of the same genotype at P < .05, Kruskal–Wallis ANOVA on ranks test with Dunn's method), the data for each sex were analyzed separately. ∗Different from BL in HbSS(nh) mice of both sexes at P < .001 (F[4,132] = 15.2) and #different from HbAA mice in both sexes at P < .001 (F[3,132] = 18.2), 2-way repeated-measures ANOVA with Bonferroni t test, n = 6 to 12 mice/group. (D) Cold-induced hyperalgesia was accompanied by an increase in heart rate in HbSS mice (F[1,44] = 10.5, P = .008, 2-way repeated-measures ANOVA). HbAA mice had no changes in heart rate (P = 1.0). ∗Different from BL at P < .01 and #different from HbAA mice at P < .01, Bonferroni t test, n = 6 to 7 mice/group. (E) Exposure to cold produced microvascular stasis that was greater in HbSS(nh) mice. ∗Different from HbAA mice at P < .001 (F[1,24] = 240.4), 2-way repeated-measures ANOVA with Bonferroni t test, n = 4 mice/group. (F) Exposure to cold caused transient hypoxia in HbSS(nh), but not HbAA, mice. Hypoxia (hypoxemia) was defined as a decrease in SpO2 in blood. ∗Different from BL at P < .001 (F[4,44] = 9.39) and #different from HbAA mice at P = .016 (F[1,44] = 8.00), 2-way repeated-measures ANOVA with Bonferroni t test, n = 6 to 7 mice/group. (G) Exposure to cold did not produce hypothermia in HbSS or HbAA mice at any time (F[4,44] = 0.177, P = .949, 2-way repeated-measures ANOVA with Bonferroni t test, n = 6 to 7 mice/group).
Figure 2
Figure 2.
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.
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