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B.M. Lutz et al.
ia exposure, led to the attenuation of ipsilateral mechani- cal, thermal, and cold pain hypersensitivities in male HbSS mice (Figure 3 A-D), although it did not affect basal or post-hypoxia ipsilateral responses in HbAA mice (Figure 3A-D) or contralateral responses in HbAA and HbSS mice (Online Supplementary Figure S5). Similar ABT-627 efficacy was seen in male BerkSS mice after hypoxia/reoxygena- tion exposure (Online Supplementary Figure S6 A-D). There were no sex-based differences in pain hypersensitivities and ABT-627 efficacy after hypoxia/reoxygenation expo- sure (Figure 3 A-D, Online Supplementary Figure S7).
Spontaneous ongoing pain in HbSS mice using a CPP paradigm was also examined. HbSS mice, but not HbAA mice, given vehicle once a day for 4 days prior to and dur- ing CPP testing (Online Supplementary Figure S1), spent sig- nificantly more time in the lidocaine-paired chamber after conditioning compared to their pre-test time (Figure 3 E, F). When given ABT-627 systemically (i.p) once a day for four days prior to and during testing (Online Supplementary Figure S1), neither HbSS nor HbAA mice displayed a significant lidocaine-paired chamber prefer- ence (Figure 3 E, F).
Effect of knockdown of DRG ET receptors on SCD pain
specific knockdown of ETA receptors was validated using Western blotting and immunostaining (Online Supplementary Figure S8 A, B). Two months after bone mar- row transplantation, donor hemoglobin expression in the recipient mice was confirmed using isoelectric focusing (Online Supplementary Figure S8 C). Mice receiving BerkSS bone marrow only expressed human βs globin (βSS). BerkAA bone marrow recipients expressed the normal human β globin (βAA; Online Supplementary Figure S8 C) and were used as a control. Mouse hemoglobin (Hb) was not found in the blood of recipient mice (Online Supplementary Figure S8 C), confirming the knockdown of mouse globin.
Bilateral evoked pain hypersensitivities as evidenced by
the increase in PWF to mechanical stimuli and the
The effects of ABT-627 hindpaw administration
described above suggest the role of peripheral ETA recep-
tors in SCD-associated pain; however, ABT-627 lacks cell-
type specificity and may also have off-target effects. As a
complementary approach, we generated a DRG neuron-
specific ETA receptor knockout mouse by crossing an
ET flox/flox mouse with an Advillincre/+ mouse in which Cre- A
recombinase is expressed only in Advillin-positive neu- rons exclusive to the DRG and trigeminal ganglia. DRG-
mal human βAA maintained normal basal responses after bone marrow transplantation under normoxia and hypox- ia/reoxygenation conditions (Figure 4 A-D).
Increased ET-1 and ETA receptor expression in SCD DRG
Our behavioral observations indicate the significance of DRG ETA receptors in pain hypersensitivity in HbSS mice. We next examined the expression of the ETA receptor and
decrease in PWL to thermal and cold stimuli developed in
the ET flox/flox mice expressing βSS (Figure 4 A-D) 2 months A
after bone marrow transplantation. These pain hypersen- sitivities had a tendency of aggravation after hypoxia/reoxygenation (Figure 4 A-D). ET cre/flox mice
A expressing βSS failed to develop these pain hypersensitiv-
ities after bone marrow transplantation in both normoxic
and post-hypoxia/reoxygenation conditions (Figure 4 A-
D). ET flox/flox mice and ET cre/flox mice transplanted with nor- AAA
A
B
CD
Figure 1. Male Townes HbSS mice display evoked pain hypersensitivity. Paw withdrawal frequencies (PWF) to a 0.16 g low force (A) and a 0.4 g medium force (B) von Frey filaments and paw withdrawal latencies (PWL) to thermal (C) and cold (D) stimuli on both left and right hind paws of 4 month old male HbAA and HbSS mice. n = 10 mice/genotype, **P< 0.01 vs. the corresponding HbAA group.
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