Cold hypersensitivity increases with age in mice with sickle cell disease

Abstract

Sickle cell disease (SCD) is associated with acute vaso-occlusive crises that trigger painful episodes and frequently involves ongoing, chronic pain. In addition, both humans and mice with SCD experience heightened cold sensitivity. However, studies have not addressed the mechanism(s) underlying the cold sensitization or its progression with age. Here we measured thermotaxis behavior in young and aged mice with severe SCD. Sickle mice had a marked increase in cold sensitivity measured by a cold preference test. Furthermore, cold hypersensitivity worsened with advanced age. We assessed whether enhanced peripheral input contributes to the chronic cold pain behavior by recording from C fibers, many of which are cold sensitive, in skin-nerve preparations. We observed that C fibers from sickle mice displayed a shift to warmer (more sensitive) cold detection thresholds. To address mechanisms underlying the cold sensitization in primary afferent neurons, we quantified mRNA expression levels for ion channels thought to be involved in cold detection. These included the transient receptor potential melastatin 8 (Trpm8) and transient receptor potential ankyrin 1 (Trpa1) channels, as well as the 2-pore domain potassium channels, TREK-1 (Kcnk2), TREK-2 (Kcnk10), and TRAAK (Kcnk4). Surprisingly, transcript expression levels of all of these channels were comparable between sickle and control mice. We further examined transcript expression of 83 additional pain-related genes, and found increased mRNA levels for endothelin 1 and tachykinin receptor 1. These factors may contribute to hypersensitivity in sickle mice at both the afferent and behavioral levels.

Keywords: Aging; C fiber; Endothelin 1; KCNK; TRPA1; TRPM8.

Figure 1

Sickle mice display cold temperature aversion, which is enhanced following induced sickling crises. (A) Both HbAA and HbSS male mice show no preference between the sides of the thermal preference plate at baseline when both sides are at 30 degrees (“baseline”), as evidenced by both groups spending time on one side at a rate not significantly different from chance during a 5 minute test (50%, P > 0.05). When the temperature on the test plate was reduced to 23°C, HbSS mice spent significantly less time on the cold plate compared to their baseline preferences (**P < 0.01). Following hypoxia and reoxygenation, sickle animals showed an enhanced avoidance of the cold plate compared to the control animals, when tested on cold plates of both 23°C (* P < 0.05) and 20°C (* P < 0.05). (B) During all behavioral thermal preference tests, there were no differences in the number of plate crosses compared between sickle and HbAA control animals (P > 0.05).

Figure 2

C fibers from sickle mice are sensitized to cold stimuli. (A) C fibers isolated from sickle mice were activated by cold stimulation at a similar proportion compared to HbAA control C fibers. (B) Cold-activated C fibers from sickle mice had a significantly lower cold detection threshold, meaning they responded to cold stimulation at warmer temperatures than HbAA controls (P < 0.05). (C) There was no significant difference in the number of action potentials fired by cold-responsive C fibers from SCD or HbAA mice in response to cold stimulation (P = 0.1848).

Figure 3

Cold aversion increases with age in sickle mice. Aged mice are on average 18.4 (± 0.4) months; younger adult mice are on average 7.9 (± 0.3) months. Mouse behavior was tested using a thermally-controlled floor with one side at 30°C and the other at 23°C, and cold aversion was measured as the percent of time spent on the colder plate during a 5 minute test. (A) Aged male HbSS mice portrayed heightened cold aversion compared with younger adult HbSS mice (*** P < 0.001). Additionally, the aged HbSS mice showed much greater cold aversion than their aged control counterparts (**** P < 0.0001). Aged male HbAA mice (controls) show no difference in cold aversion or thermal preference compared to younger adult HbAA mice (P > 0.05). (B) There were no differences in the number of crosses performed by older control or HbSS mice (P > 0.05).

Figure 4

Sickle and control (C57) DRGs have similar functional expression of cold-sensitive ion channels. (A) Isolated small-diameter DRG neurons from control (C57BL/6) and sickle mice were exposed to 100 μM menthol, and their responses assessed via calcium imaging. A similar percentage of small neurons from control and sickle mice responded to the chemical stimulation. (B) Control (C57) and sickle neurons responded to 100 μM menthol with a similar amplitude response of calcium influx, as measured by percent increase in Fura-2 ratio over baseline measurements. (C) Additional groups of small-diameter DRG neurons were exposed to 70 μM cinnamaldehyde. There was no significant difference in the percentage of sickle or control neurons responding to cinnamaldehyde stimulation. (D) Control and sickle neurons also responded to 70 μM cinnamaldehyde stimulation with similar amplitude increases in intracellular calcium.

Figure 5

Sickle (HbSS) and HbAA control DRGs have similar mRNA expression levels of cold-sensitive ion channels. (A-D) There were no differences in the mRNA expression of either Trpa1 (A-B) or Trpm8 (C-D) between control and SCD DRGs obtained from aged or younger adult mice. (E) Similarly, there were no differences in the expression levels of Kcnk2, Kcnk10, or Kcnk4 measured from DRGs collected from younger adult SCD and HbAA mice (P > 0.05).