Page 47 - Haematologica July

DRG ET receptors mediate SCD pain through the activa- A
tion of NF-κB and subsequent increase of Nav1.8 expres- sion in the DRG.
Methods
Animals
All procedures used in this study have been approved by the Rutgers New Jersey Medical School Animal Care and Use Committee and adhere to the ethical guidelines of the National Institutes of Health and the International Association for the Study of Pain. All experiments were designed to minimize animal suffer- ing and the number of animals used, and were conducted in a blind manner. Detailed animal information can be found in Online Supplementary Methods.
Behavioral analysis
Mechanical testing, thermal testing, and cold testing were con- ducted on the same day with at least 1 hour rest periods between tests as described.22 The conditioned place-preference (CPP) test was carried out as described22 and in the Online Supplementary Methods.
Hypoxia/Reoxygenation
Hypoxia/reoxygenation exposure for the induction of vaso- occlusion was conducted in the same manner as previously described.20 The detailed procedures can be found in the Online Supplementary Methods.
Genetic Knockdown and Bone Marrow Transplantation
Chimeric mouse generation was completed using a previously described protocol, which showed the development of the sickle cell phenotype in BerkSS bone marrow-recipient mice beginning at 2 months after transplantation.23 The detailed procedures can be found in the Online Supplementary Methods.
Isoelectric focusing, cell culture, immunofluorescence, Western blotting, qRT-PCR, ChIP assay, luciferase assay, electrophysiolog- ical recording, and statistical analysis methods can be found in the Online Supplementary Methods.
haematologica | 2018; 103(7)
Mouse model of sickle cell disease
In SCD patients, blood plasma ET-1 levels are elevated at a basal level and increase further during an acute vaso- occlusive crisis.13,14 Elevated ET-1 and its subsequent acti- vation of ETA receptors have been demonstrated to con- tribute to SCD pathology, including renal injury15 and pulmonary hypertension.16 The level of ET-1 mRNA is also increased in the DRG of SCD mice.17 However, whether and how this elevated ET-1 in SCD DRG con- tributes to SCD-associated pain remains elusive. Given that ETA receptor antagonists have been used in phase II/III clinical trials for cancer treatment18,19 and SCD-relat- ed pulmonary hypertension,16 identifying the role of ET- 1 and its ETA receptors in SCD-associated pain may pro- vide a new avenue for treatment of SCD-associated pain in patients.
Humanized mouse models of SCD display persistent
pain hypersensitivity that can worsen with exposure to
hypoxia.17,20,21 In the present study, using the humanized
Townes (HbSS) and Berkeley (BerkSS) mouse models of
SCD, we first examined whether local pharmacologic
inhibition or conditional knockout of ETA receptors in
DRG neurons affected pain hypersensitivity in SCD mice.
We then examined whether SCD mice expressed elevated
levels of ET-1 and ETA receptors in DRG neurons. Finally,
we unveiled a possible molecular mechanism by which
Results
Pain hypersensitivity in SCD mouse models
We first characterized basal pain behaviors in our colony of Townes humanized SCD mice. HbSS and HbAA mice (as a control) aged 4-6 months were used. Like male BerkSS mice (Online Supplementary Figure S2A, B), male HbSS mice displayed significant bilateral increas- es in basal paw withdrawal frequencies (PWF) in response to 0.16 g and 0.4 g von Frey filaments (Figure 1 A, B), and reductions in paw withdrawal latencies (PWL) to both thermal and cold stimuli when compared to their HbAA littermates (Figure 1 C, D). Similar pain hypersensitivities were seen in female HbSS mice (Online Supplementary Figure S3 A-D).
We then examined whether pain hypersensitivities stemmed from an increase in DRG neuronal excitability in HbSS mice. Compared to HbAA mice, the medium and small DRG neurons of HbSS mice showed increases of 9.85 mV and 8.24 mV, respectively, in the resting mem- brane potentials (Figure 2A) and decreases of 36% and 52%, respectively, in the current thresholds for action potential generation (Figure 2B). The number of action potentials evoked by a stimulation of ≥ 200 pA in medium and small neurons was significantly higher in HbSS mice (Figure 2C, D), although both mice displayed similar membrane input resistances and other action potential parameters, (Online Supplementary Table S1). In addition, a larger percentage of small and medium HbSS DRG neu- rons exhibited spontaneous activity compared to HbAA DRG neurons (Figure 2 E, F). The frequency of sponta- neous action potential generation was also higher in HbSS small and medium DRG neurons compared to HbAA neu- rons (Figure 2 G). These indicators of heightened excitabil- ity were not seen in large DRG neurons (Figure 2 A-G).
Effect of DRG ETA receptor inhibition on SCD pain
To address the role of DRG ETA receptors in SCD-asso- ciated pain, we subcutaneously (s.c.) injected ABT-627, a selective and specific ETA receptor antagonist,24 into the plantar side of the left hindpaw. Single administration of 5 nmol ABT-627, 2 hours before behavioral testing under normoxic conditions, led to the attenuation of ipsilateral mechanical (Figure 3A, B; Online Supplementary Figure S3A, B), thermal (Figure 3C; Online Supplementary Figure S3C) and cold (Figure 3D; Online Supplementary Figure S3D) pain hypersensitivities in the male and female HbSS mice. This effect is dose-dependent (Online Supplementary Figure S4). ABT-627 did not alter the basal mechanical, thermal and cold responses of the male and female HbAA mice (Figure 3 A-D; Online Supplementary Figure S3 A-D). Similar ABT- 627 efficacy was evident in male BerkSS mice (Online Supplementary Figure S2 A-D).
Exposure to hypoxia/reoxygenation in SCD mice has been implicated as a method of inducing vaso-occlusion and mimicking acute painful episodes in SCD patients.20 We further performed behavior analyses following hypox- ia/reoxygenation exposure in SCD mice. Male HbSS mice exhibited similar responses to the 0.4 g von Frey filament (Figure 3B), thermal (Figure 3C), and cold (Figure 3D) stim- uli before and after hypoxia/reoxygenation. However, after hypoxia/reoxygenation, bilateral PWF to the 0.16 g von Frey filament increased by 20% in male HbSS mice compared to basal values (Figure 3A). ABT-627 adminis- tered to the unilateral hind paw immediately after hypox-
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