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

TO THE EDITOR:

Sickle cell disease (SCD) is the most common hemoglobinopathy and is caused by a point mutation in the β-globin chain that is susceptible to polymerization, giving red blood cells (RBCs) a rigid sickle shape and causing vaso-occlusion.¹ Several factors enhance vaso-occlusion,^2,3 which lead to a vaso-occlusive crisis (VOC) that is accompanied by severe acute pain often requiring hospitalization and opioid treatment.⁴ Because pain itself maintains VOC,^2,3 effective pain management is of particular importance for the relief of VOC. To better understand the mechanisms underlying acute pain during VOC, a model is needed that mirrors the clinical features of VOC. Considering that low ambient temperature can be a trigger for painful VOC,³ we developed a model of acute painful VOC caused by exposure to cold in mice with SCD. We investigated whether exposure to cold causes acute pain in mice with SCD and whether this reflects the clinical features of VOC.

Male and female transgenic Berkeley mice⁵ (5-9 months old) that express human sickle hemoglobin S (HbSS) or normal human hemoglobin A (HbAA) were used. Mechanical hyperalgesia was defined as a decrease in paw withdrawal threshold using calibrated von Frey monofilaments according to the up-down method.⁶ Heat hyperalgesia was defined as a decrease in paw withdrawal latency in response to a radiant heat stimulus.^7,8 Deep tissue hyperalgesia was defined as a decrease in grip force measured by a grip force meter.^7,9 Baseline (BL) measurements were taken over 3 days before each experiment. Paw withdrawal threshold and paw withdrawal latency were measured for both hind paws and averaged. Different modalities of hyperalgesia were studied in different groups of mice. HbSS mice that exhibited BL mechanical threshold and withdrawal latency less than 2 standard deviations (SDs) and grip force less than 1 SD of the average value for HbAA mice were considered nonhyperalgesic HbSS (HbSS[nh]). Consistent with our previous report,¹⁰ the number of HbSS(nh) mice was ∼15% of all HbSS mice, and this decreased with age. HbSS(nh) mice were exposed to cold to evoke acute hyperalgesia, an experimental analogue of pain in patients.

For exposure to cold, mice were placed in a plastic cage without bedding in a room with a controlled ambient temperature of 10°C (50°F) for 1 hour and then returned to room temperature (22°C). Hyperalgesia and measures of blood oxygen saturation (SpO₂) were determined before, immediately after, and 1, 2, and 24 hours after exposure to cold. Blood SpO₂ was determined using a pulse oximeter (Kent Scientific, Torrington, CT) applied to the hind paw with simultaneous measurement of heart rate and core temperature in anesthetized mice (1.8%-2% isoflurane). A decrease in SpO₂ in blood was an indication of hypoxia (hypoxemia). Stasis measurements were performed in 20 to 25 flowing venules through implanted dorsal skin fold chambers.¹¹ The same venules were reexamined for stasis (no flow) every hour for 4 hours after exposure to cold. Percent of static venules was calculated as static venules/total venules × 100. For all experiments, the experimenter was blind to the genotype and treatment.

Blood collection and isolation of plasma and blood cells¹⁰ were performed at 2 hours after exposure to cold. The levels of cyclooxygenase-2 (COX-2) and diacylglycerol lipase β (DAGLβ) proteins in blood cell lysates were determined by western blotting (45 μg of protein per sample), defined as the density of the immunoreactivity of the protein of interest/total protein within each sample (Revert 700 Total Protein Stain, LI-COR) and expressed as a percent of the corresponding average amount in HbAA mice. Specificity of the DAGLβ and COX-2 antibodies was confirmed.¹⁰ The level of endocannabinoid 2-arachidonoylglycerol (2-AG) was analyzed in plasma by liquid chromatography with tandem mass spectrometer and quantified against deuterated internal standard.¹⁰ 

Data are presented as mean ± standard error of the mean and were analyzed by 1- or 2-way analysis of variance (ANOVA) with repeated measures followed by Bonferroni’s post hoc test. P < .05 was considered significant.

Because pain associated with VOC can be severe, we first determined if exposure to cold produced acute mechanical, heat, and deep tissue hyperalgesia. Unlike in HbAA mice, mechanical, heat, and deep tissue hyperalgesia developed in nonhyperalgesic HbSS mice (Figure 1A-C). Mechanical and deep tissue hyperalgesia persisted for 24 hours. In some mice, hyperalgesia did not disappear during the entire test period, which may indicate a possible transition from acute to chronic hyperalgesia. Because there were no sex differences in mechanical and heat hyperalgesia at any time, data for male and female mice were combined. BL measures of grip force were lower in female mice compared with male mice in both groups. Therefore, data for each sex were analyzed separately. Exposure to cold caused deep tissue hyperalgesia in both sexes. Hyperalgesia was accompanied by an increase in heart rate in HbSS mice, but not in HbAA mice (Figure 1D), suggesting the presence of pain-related stress.^2,3,12,13 

Exposure to cold produced vascular stasis in both HbSS and HbAA mice; however, the magnitude and duration of stasis were greater in HbSS mice (Figure 1E). Because changes in peripheral blood flow are mediated by the autonomic nervous system, it is likely that autonomic dysregulation, also observed in patients with SCD,^3,13 contributes to the increased and prolonged stasis in HbSS mice. We have shown in earlier studies that stasis caused by hypoxia-reoxygenation in sickle mice is characterized by vaso-constriction, endothelial cell activation, inflammation, and leukocyte adhesion to the vessel wall.^11,14 These factors contribute to the increase in the transit time of RBCs through capillary beds, prolonging RBC exposure to hypoxia, measured as a decrease in SpO₂ (Figure 1F). A high incidence of hypoxia has been reported among patients with SCD, which increases significantly during VOC.¹⁵ Additionally, no changes in core temperature were observed after exposure to cold in both groups of mice (Figure 1G).

Recently, we showed that an increased level of the endocannabinoid 2-AG in plasma due to an increase in its synthesizing enzyme DAGLβ in blood cells contributes to chronic hyperalgesia in HbSS mice.¹⁰ Likewise, acute hyperalgesia after exposure to cold was accompanied by an increase in these indicators in HbSS mice (Figure 2A-C). An increase in DAGLβ protein may be associated with an overall increase in the number of immune cells^16,17 expressing DAGLβ.^18,19 2-AG is oxidized by COX-2 to form pronociceptive derivatives, and high levels of COX-2 were observed in the blood cells of HbSS mice regardless of hyperalgesia status¹⁰ and exposure to cold (Figure 2D). We postulate that DAGLβ-mediated accumulation of 2-AG in the presence of high COX-2 level potentiates the formation of pronociceptive metabolites that contribute to acute hyperalgesia.^10,20 Administration of KT109, which blocks DAGLβ activity,^18,21 1 hour before exposure to cold blocked the development of mechanical hyperalgesia (Figure 2E).

We have developed a model of cold-induced VOC similar to that in patients with SCD to investigate the mechanisms underlying the induction of VOC and associated pain to develop effective approaches for prevention. Because elevated levels of 2-AG contribute to both acute and chronic hyperalgesia, controlling the level of this endocannabinoid may be a promising target for pain relief in SCD.