Descending Facilitation of Nociceptive Transmission From the Rostral Ventromedial Medulla Contributes to Hyperalgesia in Mice with Sickle Cell Disease

Abstract

Sickle cell disease (SCD) is an inherited blood disorder that is associated with acute episodic and chronic pain. Mice with SCD have robust hyperalgesia mediated, in part, by sensitization of spinal dorsal horn neurons. However, underlying mechanisms are not fully understood. Since the rostral ventromedial medulla (RVM) is a major component of descending circuitry that modulates nociceptive transmission in the spinal cord, we examined if the RVM contributes to hyperalgesia in mice with SCD. Injection of lidocaine, but not vehicle, into the RVM eliminated mechanical and heat hyperalgesia in sickle (HbSS-BERK) mice without altering mechanical and heat sensitivity in naïve C57B mice. These data indicate that the RVM contributes to the maintenance of hyperalgesia in mice with SCD. In electrophysiological studies, we determined the changes in response properties of RVM neurons that might contribute to hyperalgesia in sickle mice. Recordings were made from single ON, OFF, and Neutral cells in the RVM of sickle and control (HbAA-BERK) mice. Spontaneous activity and responses of ON, OFF and Neutral cells evoked by heat (50 °C) and mechanical (26 g) stimuli applied to the hind paw were compared between sickle and control mice. Although there were no differences in the proportions of functionally-identified neurons or spontaneous activity between sickle and control mice, evoked responses of ON cells to heat and mechanical stimuli were increased approximately 3-fold in sickle mice as compared to control mice. Thus, the RVM contributes to hyperalgesia in sickle mice via a specific ON cell-dependent descending facilitation of nociceptive transmission.

Keywords: electrophysiology; hyperalgesia; mouse; rostral ventromedial medulla; sickle cell disease.

Figure 1.

Effects of the lidocaine injection into the RVM of sickle and naïve C57B mice. A: Histological verification of the location of injecting sites in sickle (black dots) and C57B (white dots). The gray rectangles represent the area of the RVM according to our previous stereological studies (Khasabov and Simone 2013). Coordinates from bregma according to (Paxinos and Franklin 2001) are provided at the left. Structures are indicated in sections of the brainstem: 7N, facial nucleus; 7n, facial nerve; Amb; ambiguous nucleus; py, pyramidal tract; sp5, spinal trigeminal tract; IRt, intermediate reticular nucleus. B: Confirmation of injection into the RVM by the localization of microspheres (red) at the injecting site marked in A by the arrowhead. Nuclei are constrained by DAPI (blue). a) the tip of the guide cannula; b) the tip of the injection cannula; c) distribution of 0.2 μl bolus of 50% microsphere suspension. Lidocaine but not vehicle decreased mechanical (C) and heat hyperalgesia (D) in the sickle mice (red dots). In C57B mice both types of injections were without effects (blue dots). 0 refers to baseline values obtained before RVM injections. Statistically significant differences are indicated by: *difference compared to corresponding baseline * p<0.05, **p<0.001; # difference between groups injected with lidocaine, p<0.001; ^difference between groups injected with vehicle, p<0.001. Two Way Repeated Measures ANOVA followed by Bonferroni t-tests.

Figure 2.

Histological verification of the location of recording sites in control (AA) and sickle (SS) mice. Same anatomical landmarks as in Figure 1. ON cells are denoted by squares, OFF cell by triangles, and Neutral cells by circles. Recording sites in red represent those from female mice whereas those in blue represent recording sites in male mice.

Figure 3.

Responses of ON cells to heat. A: representative examples of responses of ON cells in sickle (SS) and control (AA) mice. ON cells in sickle mice showed clear sensitization to heat stimuli. This was evidenced by an increased firing rate at lower temperature (lower response threshold), as well as greater discharge frequency and duration of response as compared to control mice. The accompanying EMG response threshold was also lower in sickle mice compared to control mice. B: mean (±SEM) heat response thresholds for neurons was lower in sickle than control mice. C: mean (±SEM) number of impulses evoked by the heat stimulus was higher in sickle mice than control mice. D: mean (±SEM) duration of response was longer in sickle mice than in control mice. Method for determining response threshold for neurons is illustrated by dashed lines: red indicates sickle mice, blue indicates control mice. Thresholds of EMG responses are indicated by dotted lines, which point to the EMG response: red is for sickle and blue is for control mice. Asterisks indicate differences between groups, ** p<0.01, *** p<0.005.

Figure 4.

Responses of OFF cells to heat. A: representative examples of responses of OFF cells in sickle (SS) and control (AA) mice. OFF cells responses, which were expressed as a pause in discharge rate following noxious stimulation, were similar in sickle and control mice. However, the accompanying EMG response threshold was lower in sickle mice compared to control mice. Thresholds of EMG responses are indicated by dotted lines, which point to the EMG response: red is for sickle and blue is for control mice. B, C, D: mean (±SEM) heat response thresholds, number of impulses evoked by heat stimulation, and response duration, respectively, were not differ between groups. All designations of straight lines are as in Figure 2.

Figure 5.

Representative examples of Neutral cells in sickle (SS) and control (AA) mice. Heat stimulation of the paw did not produce any neuronal responses, however, the accompanied EMG response in the sickle mouse was characterized by lower threshold compared to the control mouse. Thresholds of EMG responses are indicated by dotted lines, which point to the EMG response: red is for sickle and blue is for control mice.

Figure 6.

Responses of ON and OFF cells to mechanical stimuli. A: Representative examples of responses of ON cells to mechanical stimulation with the 26g von Frey monofilament (5 sec) in sickle (SS) and in control (AA) mice. B: mean (±SEM) numbers of impulses evoked in ON cells by mechanical stimuli in SS and AA mice. C: Representative examples of responses of OFF cells evoked by mechanical stimuli in SS and AA mice. D: mean (±SEM) numbers of impulses evoked by the von Frey monofilament in OFF cells did not differ between groups. Horizontal bars in A and C indicate the time of stimulation. Asterisks indicate a significant difference between groups: **** p<0.001.