Mouse models for studying pain in sickle disease: effects of strain, age, and acuteness

Abstract

The clinical management of severe pain associated with sickle cell disease (SCD) remains challenging. Development of an optimal therapy would be facilitated by use of murine model(s) with varying degrees of sickling and pain tests that are most sensitive to vaso-occlusion. We found that young (≤3 months old) NY1DD and S+S(Antilles) mice (having modest and moderate sickle phenotype, respectively) exhibited evidence of deep tissue/musculoskeletal pain. Deep tissue pain and cold sensitivity in S+S(Antilles) mice increased significantly with both age and incitement of hypoxia/reoxygenation (H/R). C57/BL6 mice (genetic background strain of NY1DD and S+S(Antilles) ) were hypersensitive to mechanical and heat stimuli, even without the sickle transgene. H/R treatment of HbSS-BERK mice with severe sickle phenotype resulted in significantly decreased withdrawal thresholds and enhanced mechanical, thermal and deep tissue hyperalgesia. Deep hyperalgesia incited by H/R in HbSS-BERK was ameliorated by CP 55940, a cannabinoid receptor agonist. Thus, assessment of deep tissue pain appears to be the most sensitive measure for studying pain mechanisms across mouse models of SCD, and HbSS-BERK mice may be the best model for vaso-occlusive and chronic pain of SCD.

Figure 1. Pain behaviors in young and old NY1DD and S+S^Antilles and HbSS-BERK mice

Sensitivity to nociceptive stimuli is shown in different groups of mice comprising, young and old, NY1DD and S+S^Antilles mice (sickle mice) and their age-matched control C57/BL6 mice; and HbSS-BERK (severe sickle phenotype) and their age-matched control HbAA-BERK mice. Mean age in months ± SEM for, ‘Young’ mice: C57/BL6, 2.2 ± 0.2; NY1DD, 2.6 ± 0.2; S+S^Antilles, 3.0 ± 0.0, and ‘Old’ mice, C57/BL6, 6.5 ± 0.0; NY1DD, 5.3 ± 0.2; S+S^Antilles, 7.8 ± 0.8; HbAA-BERK (control), 5.5 + 0.5 and HbSS-BERK, 5.7 + 0.7. Comparisons were made between age-matched, NY1DD and C57/BL6; between S+S^Antilles and C57/BL6; and S+S^Antilles and NY1DD and differences in nociceptive sensitivity due to age were also compared between ‘young’ and ‘old’ mice (A–F). (A) Mechanical threshold using von Frey filaments, measure of cutaneous nociception is shown. (B) Paw withdrawal frequency (PWF) in response to 10 applications of a 9.8 mN (1.0 g) von Frey monofilament in the same mice. A higher PWF indicates increased nociception. No statistically significant difference was observed between age-matched sickle and C57/BL6 mice or between younger and older mice. (C) Grip force measurements indicating deep tissue/musculoskeletal pain are shown. Lower grip force suggests increased deep tissue hyperalgesia. Both types of sickle mice show decreased grip force compared to age-matched C57/BL6 ‘young’ NY1DD mice compared to ‘young’ S+S^Antilles mice and ‘old’ S+SAntilles show decreased grip force as compared to ‘young’ S+S^Antilles. (D) Paw withdrawal latency (PWL) on the cold plate in ‘seconds’ when the mouse lifted its paw after being placed on the cold plate maintained at 4°C, is shown. ‘Old’ S+S^Antilles mice show significantly decreased PWL (increased cold sensitivity) as compared to age-matched NY1DD and C57/BL6 and ‘young’ S+S^Antilles mice. (E) Paw withdrawal frequency (PWF) on a cold plate at 4°C in a 2 min period is shown. Old S+S^Antilles mice demonstrate significantly higher PWF (increased cold sensitivity) than young S+S^Antilles, and age-matched C67/BL6 and NY1DD. (F) PWL in seconds in response to a heat stimulus targeted to the intraplantar surface of the hind paw is shown. Of note, NY1DD show significantly less sensitivity to heat (higher PWL). (G–L) Sensitivity to different nociceptive stimuli in HbSS-BERK (sickle) as compared to HbAA-BERK. Please note that HbSS-BERK show significantly high sensitivity to all nociceptive stimuli as compared to HbAA-BERK. Significance (A–L), *P<.05, **P<.01 and ***P<.001, Each data point is the mean + SEM of measurements from 7–13 different mice. BW, body weight.

Figure 2. Comparisons of pain behaviors between different strains of control mice

Columns in each graph represent the mean ± SEM of C57/BL6 (left) and HbAA-BERK (right) mice. (A) Mechanical withdrawal thresholds of C57/BL6 mice were significantly lower (*P = .0012), i.e., more sensitive, than HbAA-BERK mice. (B) No differences were observed between the two types of mice for PWF to von Frey filaments. (C) Weight-normalized grip force. (D) PWL on a cold plate maintained at 4°C. (E) PWF on a cold plate at 4°C over a 2 min period. (F) PWL to heat was lower (more sensitive) in C57/BL6 compared to HbAA-BERK mice (^$P = .044). Mouse ages (mean ± SEM): C57/BL6 = 6.0 months ± 0.6; HbAA-BERK mice = 5.5 months ± 0.5. n= 7–10 mice of each type. Statistical analyses were conducted using unpaired t-tests. BW, body weight; PWL, paw withdrawal latency; PWF, paw withdrawal frequency.

Figure 3. Hypoxia/reoxygenation-induced pain behaviors in sickle mice.

Following baseline pain measurements, mice were exposed to first hypoxia treatment of 3h, followed by pain measures after 1–2h and 18h of re-oxygenation (HR1). This was repeated after 24h, followed by pain measures after 1–2h, 18h and 7d of reoxygenation (HR2). Different measures of pain are shown for sickle, S+S^Antilles and HbSS-BERK and their control C57/BL6 and HbAA-BERK, respectively. Data are shown as mean ± SEM. Comparisons were made between control and sickle mice following each treatment and significance is indicated with [*] sign. Within each mouse changes in nociception following treatments were compared to baseline (BL) and significance is shown with the # sign. (A and B) Paw withdrawal threshold to von Frey monofilaments are shown. Lower thresholds are indicative of increased sensitivity to cutaneous nociception. HbSS-BERK compared to HbAA-BERK: *P<.05, **P<.01, ***P<.001, ****P<.0001; significant differences from initial baseline value: ^#P<.05. (C and D). PWF with 9.8 mN (1.0 g) von Frey monofilament is shown. Higher PWF indicates increased nociception. In (C), *P<.01, **P<.001, ***P<.0001, HbSS-BERK compared to HbAA-BERK; ^#P<.05, compared to baseline (D) ^#P<.05 and ^##P<.001, compared to baseline in C57/BL6 and S+S^Antilles. (E and F) Grip force measurements indicative of deep tissue/musculoskeletal pain are shown. Lower grip force is suggestive of increased deep pain. In (E) *P<.05, **P<.001, ***P<.0001 as compared to HbAA-BERK and #P<.05, compared to baseline. In (F) *P<.05, compared to C57/BL6 and ^#P<.05 and ^##P<.001 compared to baseline. (G and H) Paw withdrawal latency (PWL) on the cold plate in ‘seconds’ when the mouse lifted its paw after being placed on the cold plate maintained at 4°C, is shown. In (G) *P<.05, **P<.01, ***P<.0001, compared to HbAA-BERK and ^#P<.05, as compared to baseline. In (H) *P<.05, as compared to C57/BL6 and ^#P<.05, ^##P <.01 compared to baseline. (I and J) Paw withdrawal frequency (PWF) on a cold plate at 4°C in a 2 min period is shown. In (I) *P<.01, **P<.001, compared to HbAA-BERK and ^#P<.01, ^##P<.001, compared to baseline. In (J) *P<.05, **P<.01, compared to C57/BL6; ^#P<.001 compared to baseline. (K and L) PWL in seconds in response to a heat stimulus targeted to the intraplantar surface of the hind paw is shown. Shorter latency indicates increased sensitivity to heat. In (K) *P<.0001, compared to HbAA-BERK and ^#P<.05, ^##P<.01, ^###P<.001 compared to baseline. In (L) *P<.01, **P<.0001 compared to C57/BL6. All data were obtained from 5.7 ± 0.7 mo old HbSS-BERK (open squares), 5.5 ± 0.5 mo old HbAA-BERK (solid squares), 5.6 ± 0.1 mo old S+S^Antilles (open triangles) and 6.0 ± 0.6 mo old C57/BL6 (solid triangles) mice. N = 7–10 per type of mice. BW, body weight.

Figure 4

The cannabinoid receptor agonist CP 55940 reduces deep hyperalgesia following hypoxia reoxygenation. Following baseline grip force measurements, HbSS-BERK mice were exposed to 3h of hypoxia treatment and 1h of oxygenation at room air (H/R), and grip force was measured again. Mice were then injected with vehicle or CP 55940 and grip force was measured at indicated time on the graph. Hollow and solid circles represent vehicle and CP 55940 treatment, respectively. Data are shown as mean + SEM from 5 mice per treatment. Statistical significance was calculated by comparing each value with BL ($) or with H/R (*); and between vehicle and CP 55940 treatment (#). ^$P<0.05 and ^$$P<0.005, as compared to BL; *P<0.05 and **P<0.005 as compared to H/R; and ^#P<0.05 and ^##P<0.005, as compared to vehicle for that time point. Mean age of mice + SEM in months were, Vehicle, 12.25 + 0.8 and CP 55940, 11.7 + 0.7. Abbreviations, BL, baseline; H/R, hypoxia reoxygenation; Veh, vehicle; CP, CP 55940.