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Published in final edited form as: Neuroscience. 2023 Jun 16;526:1–12. doi: 10.1016/j.neuroscience.2023.06.007

Descending facilitation of nociceptive transmission from the rostral ventromedial medulla contributes to hyperalgesia in mice with sickle cell disease

Victoria M Rogness 1, Joseph Juliette 1, Iryna A Khasabova 1, Kalpna Gupta 2, Sergey G Khasabov 1, Donald A Simone 1
PMCID: PMC10528639  NIHMSID: NIHMS1918624  PMID: 37330194

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: Sickle cell disease, mouse, hyperalgesia, rostral ventromedial medulla, electrophysiology

INTRODUCTION

Sickle cell disease (SCD) is the most commonly inherited blood disorder and results from a point mutation in the β-globin gene, causing polymerization of the hemoglobin S subunits and distorting red blood cells (RBCs) into a crescent shape (Ingram, 1956; Noguchi and Schechter, 1981). SCD is associated with vaso-occlusion, vasculopathy, endothelial activation, hemolysis, inflammation, oxidative stress and ischemiareperfusion injury (Hebbel et al., 2004; Kassim and DeBaun, 2013). Pain, both acute and chronic, is a characteristic feature of SCD (Ballas and Gupta, 2012). Hemolysis, oxidative stress, inflammation, vaso-occlusion, vasculopathy, ischemia-reperfusion injury, organ damage, and neuropathy may contribute to chronic pain in SCD (Lei et al., 2021; Sagi et al., 2021; Tran et al., 2021). Quantitative sensory testing in patients showed that chronic pain includes ongoing pain and increased cutaneous sensitivity to mechanical, heat, and cold stimuli (Bakshi et al., 2018; Brandow and Panepinto, 2016; Brandow et al., 2013; Jacob et al., 2015), and evidence of central sensitization (Campbell et al., 2016; Darbari et al., 2015). Opioids remain the primary treatment for pain in SCD (Ballas, 2007) despite their serious side effects.

Transgenic, humanized murine models of SCD, including the Berkeley model (Pástzy et al., 1997), are used to study pain mechanisms in SCD. HbSS-BERK mice are homozygous for knockouts of both murine α and β globins and carry a single copy of the linked transgenes for human α and βS globins. Similar to patients with SCD, these mice exhibit chronic inflammation (Belcher et al., 2003; Hebbel et al., 2004) and hyperalgesia (Cain et al., 2012; Hillery et al., 2011; Kohli et al., 2010; Sagi et al., 2018). Cutaneous nociceptors (Hillery et al., 2011; Uhelski et al., 2017) and dorsal horn neurons (Cataldo et al., 2015) were sensitized in HbSS-BERK mice, characterized by increased spontaneous activity and enhanced responses to mechanical, heat, and cold stimuli. The mechanisms that contribute to the sensitization of dorsal horn neurons in sickle mice are not well understood.

Neurons in the rostral ventromedial medulla (RVM) project to the spinal cord and modulate nociceptive transmission (Chen and Heinricher, 2019; Heinricher et al., 2009; Mason, 1999; Millan, 2002; Ossipov et al., 2014; Porreca et al., 2002; Qing-Ping and Nakai, 1994). In the RVM, nociceptive-facilitating ON cells are excited by noxious stimuli and are characterized by a burst-like increase in their discharge in connection with a nociceptive withdrawal reflex. Activity of OFF cells is thought to inhibit nociceptive transmission and respond with a pause in their activity during noxious stimulation (Fields et al., 1983a and 1983b). ON cells are inhibited by opioids whereas OFF cells are excited (Basbaum and Fields, 1984; Fields, 2000; Fields et al., 1991; Heinricher et al., 1994). Neutral cells do not exhibit a change in discharge during noxious stimulation and their role in descending modulation is unclear. Increased responses of ON cells and an increase in the pause of OFF cell activity following intraplantar injection of capsaicin (Brink et al., 2012) and spinal nerve ligation (Carlson et al., 2007) indicate that these types of pain are characterized by parallel activation of descending facilitation and reduction of descending antinociception. In a model of inflammatory pain, hyperalgesia was associated primarily with an increase in the sensitivity of ON cells (Khasabov et al., 2012), indicating that increased descending facilitation of nociceptive transmission alone may contribute to inflammatory pain.

We observed that stimulation of serotonin release in the RVM increased serotonin in the dorsal horn of the spinal cord and reduced hyperalgesia in sickle mice (Tran et a., 2021), suggesting that the RVM might contribute to hyperalgesia in SCD. To assess the role of the RVM in hyperalgesia in sickle mice, we determined the effects of microinjection of lidocaine into the RVM on hyperalgesia in sickle mice and compared electrophysiological responses of neurons in the RVM between sickle and control mice to identify changes in activity of RVM cells that may contribute to hyperalgesia in SCD. Results indicate that the RVM maintains hyperalgesia in sickle mice through increased descending facilitation of nociceptive transmission.

MATERIALS AND METHODS

Mice

Adult (6–12 months, weighing 20–40 g) HbSS-BERK sickle mice (n=12, 10 male and 4 female), sex-matched HbAA-BERK control mice (control; n=10, 6 male and 4 female), and naïve C57B mice (n=6; 3 male and 3 female) were used (hereafter referred to sickle and control mice, and C57B, respectively). HbSS mice are homozygous for sickle hemoglobin and express >99% human sickle hemoglobin whereas HbAA mice (control mice) express normal human hemoglobin A. Neither HbSS nor HbAA mice express murine alpha- and beta-globins. Mice were bred and phenotyped as described (Sagi et al, 2018) in an AAALAC-approved mouse colony maintained under controlled environmental conditions (12 hours light/dark cycle at 23°C) and given access to food and water ad libitum. All experiments were approved by the Institutional Animal Care and Use Committee at the University of Minnesota.

Measurement of mechanical hyperalgesia

Mice were placed on an elevated mesh platform with perforations of 1 cm2 and under a clear glass cage enclosing them. Mice were acclimated to the environment for 30 minutes prior to testing. Withdrawal responses to mechanical stimuli applied to the hind paw were obtained as previously described (Khasabov et al., 2007, 2021). Briefly, a standard von Frey monofilament with a bending force of 3.9 mN was applied to the plantar surface of each hind paw 10 times, each for 1–2 sec. Care was taken to ensure that different areas of the middle portion of the plantar paw were stimulated while avoiding the pads. Slightly different areas were stimulated to avoid potential sensitization from repeated testing. Naive mice typically withdraw from this stimulus 10–20% of the time. Mechanical hyperalgesia was defined as withdrawal frequency ≥50%.

Measurement of heat hyperalgesia

Sensitivity to heat was determined by measuring the latency of a withdrawal response to radiant heat applied to the center of the plantar surface of the hind paw as previously described (Khasabova et al., 2019). Mice were placed on an elevated glass platform and allowed to habituate for 10 min. The heat stimulus was applied 3 times on each paw and consecutive applications of radiant heat were separated by at least 3 min. Heat stimuli did not exceed a duration of 20 s to prevent tissue damage. The latency of response for each hind paw was defined as the average of the 3 trials. A significant decrease in withdrawal latency was defined as heat hyperalgesia.

Cannulation of the RVM

Sickle mice with ongoing mechanical and heat hyperalgesia, and naive C57B control mice, were anesthetized with isoflurane and placed in a stereotaxic frame. Body temperature was maintained near 37°C using a feedback-controlled electrical blanket (Harvard Apparatus, Holliston, MA). A small craniotomy was made and a guide cannula (5 mm in length, 26 gauge; P1 Technologies, Roanoke, VA) was lowered toward the medial portion of the RVM. The tip of the cannula was placed at the following stereotaxic coordinates from bregma: anterior-posterior (A-P) = 5.8 mm; media-lateral (M-L)= 0 mm; and dorsal-ventral (D-V) = 4.6 mm according to the mouse brain atlas of Paxinos and Franklin (2001). The guide cannula was secured using dental resin (Duralay, Dental, Worth, IL) adhered to two stainless steel machine screws that were screwed to the skull. A 33-gauge dummy cannula was inserted into the guide cannula to maintain patency and animals were allowed to recover for 3 days before behavioral testing. To confirm the absence of an effect of RVM cannulation on mechanical and thermal hyperalgesia, withdrawal responses to mechanical and heat stimuli were determined for 2 consecutive days after implantation and before drug administration. Only mice with stable withdrawal responses before and after cannulation were used.

Drug administration into the RVM

A volume of 0.2 μl of 8% of the local anesthetic lidocaine hydrochloride monohydrate (L5647, Sigma-Aldrich, St. Louis, MO) dissolved in saline was used to anesthetize the RVM. Injection of 0.2 μl of saline was used as a control. Injections were delivered using a 33-gauge internal cannula (P1 Technologies, Roanoke, VA) that extended 1 mm below the tip of the guide cannula. Injections into the RVM were made at the following coordinates from bregma: A-P 5.8 mm; M-L 0 mm; D-V 5.6 mm using a 1-μl microsyringe (Hamilton, Reno, NV) attached to PE-10 tubing and administered slowly over a period of 2 min. The cannula was left in position for 120 sec after injection to allow for diffusion and was then removed and replaced with the dummy cannula. On the day of the experiment, withdrawal responses to mechanical and heat stimuli were determined before and at 1, 2, 3, 4 and 24 hours after injections of vehicle or lidocaine into the RVM. Each mouse received injections of lidocaine and vehicle that were given in random order with an interval of 4 days between each of the two injections. The experimenter was blinded to the treatment.

To confirm that lidocaine and vehicle were delivered into the RVM, at the end of the experiment mice received an injection of vehicle (0.2 μl) into the RVM that contained fluorescent Polystyrene Microspheres (excitation 580 / emission 605, cat. no. F13081; Invitrogen, Eugene, OR) and were visualized histologically (Khasabov et al., 2017).

Surgical preparation for electrophysiology

Mice were anesthetized using 2.0% isoflurane and placed in a stereotaxic apparatus. Body temperature was maintained at 37°C using a feedback-controlled heating pad (Harvard Apparatus, Holliston, MA). A craniotomy was performed 6 mm caudal from bregma, at the location above the RVM. The dura was removed using forceps. Isoflurane was reduced to 0.8–1.2% during electrophysiological recordings. Needle electrodes were inserted into the left biceps femoris for electromyographic recording (EMG). Respiratory rate was continuously monitored, and isoflurane was adjusted as needed (0.8–1.2%) throughout the experiments to ensure a hind limb withdrawal response, as indicated by EMG, was present in response to noxious stimulation.

Electrophysiological recording from RVM neurons

Extracellular recordings were made from single neurons in the RVM using glass-coated carbon-fiber microelectrodes (Kation Scientific, Minneapolis, MN) that were lowered into the RVM in 3-μM steps using an electronic micromanipulator (David Kopf Instruments, Tujunga, CA). The ranges of stereotaxic coordinates for all cells studied were: AP = 5.3 to 7.2 mm from bregma; DV = 4.0 to 6.3 mm from the cranial surface; ML = −1.5 to +1.5 mm from midline. Neurons were not identified until the microelectrode had descended at least 4.0 mm from the surface of the brain, into the area of the RVM. Action potentials were observed on an oscilloscope, audiomonitored, and saved on a computer using Spike II software (Cambridge Electronics Design) for offline analyses.

Functional classification and evoked responses of RVM neurons

Once a neuron was identified, spontaneous activity was recorded for 1 minute and responses to heat stimuli were determined. The left hind paw was stimulated using a Peltier device (contact area = 1 cm2) from a base temperature of 32°C to 50°C at a rate of 1°C/sec, with temperature held at 50°C for 5 sec before returning to 32°C. For offline analysis, neuronal activity was represented as a histogram with 100-msec bin widths. Baseline SA was analyzed for 1 minute prior to heat stimulation and the mean SA and standard deviation (SD) were determined. The beginning of evoked responses of ON cells was defined as a time of the first of three consecutive bins in which the sum of impulses was ≥ the mean SA +2 SD. The end of a response was defined as the last of the three such intervals. Evoked responses of ON cells were characterized as the sum of action potentials evoked during response duration with SA obtained before stimulation subtracted from the evoked response. Similarly, the duration of OFF cell responses was characterized from the first of three bins to the to the last three consecutive bins during which the number of impulses was ≤ the mean SA +2 SD. Evoked responses of OFF cells were defined as the sum of action potentials during the period of response minus SA obtained prior to stimulation, and were always negative (decrease in the number of action potentials). Neurons that did not exhibit increases or decreases in evoked activity were classified as Neutral cells. Heat response threshold was defined as the stimulus temperature at the time of the initial 100-msec period of response. The temperature threshold of the EMG response was defined as the stimulus temperature at the time of the onset of the EMG response.

Some neurons were also tested with mechanical stimuli. A von Frey monofilament (26 g) was applied to the plantar surface of the left hind paw for a duration of 5 sec. This was repeated 3 times with a minimum of 10 sec between stimuli. Responses were defined by the difference in action potentials from the 5 sec prior to stimulation and the 5 sec during stimulation. Responses from three trials were averaged. Only clear responses that were accompanied by an EMG response were used.

Histological verification of recording sites

The recording site of the last neuron studied in each experiment was marked by passing +/−50 μA of current through the recording electrode for 30 sec at each polarity. The lesion site served as a reference site to estimate the location of other recording sites within the same electrode track. Mice were euthanized by an overdose of isoflurane and underwent cardiac perfusion with 10% formalin. Lesions sites and estimated recording sites were verified histologically in 100-μm sections and reconstructed onto outlines adapted from the stereotactic atlas of mouse brain (Paxinos and Franklin, 2001). For reconstruction of recording sites, we used similar anatomical borders of the RVM that we used previously (Khasabov and Simone, 2013). In the coronal plane, the area of the RVM was considered within the shape of a rectangle with the dorsal border at the level of most dorsal margins of facial nuclei, the ventral border at the dorsal margins of the pyramids, and vertical lateral borders at the middle distance between the lateral edge of the pyramid and the most medial edge of the facial nuclei. The rostro-caudal dimension of the RVM was considered from the caudal margins of the trapezoid body to the rostral edge of the inferior olives.

Data analyses

The effects of microinjection of vehicle or lidocaine into the RVM on withdrawal response frequencies and paw withdrawal latencies were determined using two-way ANOVA with repeated measures followed by Bonferroni t-tests for individual comparisons. For the electrophysiological studies, the proportions of ON, OFF and Neutral cells were compared between sickle and control mice using chi-squared tests. Spontaneous activity and the number of action potentials for ON, OFF and Neutral cells evoked by heat and mechanical stimuli were compared between sickle and control mice using two-tailed t-tests. A p-value of <0.05 was considered significant in all cases. Mean ± SEM values are reported unless otherwise stated.

RESULTS

The RVM contributes to hyperalgesia in sickle mice

HbSS sickle mice (n=6; 4 male and 2 female) and 6 C57B mice (n=6; 3 male and 3 female) received a separate injections of lidocaine (8%) and vehicle (saline) into the RVM in random order 4 days apart to determine if the RVM contributed to hyperalgesia in sickle mice. In preliminary experiments, injection of lidocaine into the RVM of HbAA control mice (n=2) did not alter withdrawal responses. However, we used C57 mice as a control because of the limited supply of HbAA mice and because we wanted to use the HbAA mice for the electrophysiology studies. Withdrawal response frequencies for C57 mice were similar to those of HbAA mice (Khasabova et al., 2023). Data for male and female mice were similar so they were combined. Figure 1A and B show the localization of injection into the RVM based on the distribution of the fluorescent microspheres that were injected into the RVM at the end of the experiment. All injections were made into the RVM. Figure 1C shows that injection of lidocaine into the RVM decreased mechanical hyperalgesia in sickle mice for at least 4 hours, but had no effect on withdrawal responses in naïve C57 mice. Also, injection of vehicle in sickle and C57B mice did not change withdrawal responses to mechanical stimuli. Two-way ANOVA with repeated measures indicated that there was a significant interaction between group and time after injection (F51,143 = 8.458; p<0.001). At 2 and 3 h after lidocaine, mean paw withdrawal frequency decreased to less than 50% in sickle mice, and mechanical hyperalgesia recovered by 24 h. Similarly, Figure 1D shows that lidocaine also reduced heat hyperalgesia (F51,143 = 4.212; p<0.001) during the first 4 h after injection, which returned by 24 h. Injection of vehicle in sickle mice or administration of lidocaine or vehicle in C57B mice did not alter withdrawal latencies to heat. Together, these data demonstrate that the RVM contributes to the maintenance of mechanical and heat hyperalgesia in sickle mice.

Figure 1.

Figure 1.

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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.

Electrophysiological studies of the RVM in sickle mice

HbAA control and HbSS sickle mice were screened for mechanical hyperalgesia just prior to electrophysiological recording. All sickle mice, but none of the control mice, exhibited robust mechanical hyperalgesia (≥50% withdrawal frequency). The mean (±SEM) frequencies of withdrawal for sickle (n=6) and control (n=10) mice were 70.8±1.1% and 20.0±1.7%, respectively.

Localization of recording sites in the RVM

A total of 118 neurons were studied. All neurons that were classified as ON, OFF, or Neutral were located in the RVM (Figure 2). The distribution of recording sites within the RVM were similar for sickle and control mice, and for male and female mice. Because responses of neurons in male and female mice were similar, data were combined.

Figure 2.

Figure 2.

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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.

Proportions and spontaneous activity of ON, OFF and Neutral cells in sickle and control mice

We determined the proportions of ON, OFF, and Neutral cells to see if there was a greater proportion of ON or OFF cells in sickle mice, whereby Neutral cells may have changed their functional properties. In both control and sickle mice, there were approximately equal proportions of cell types found. In control mice, 50% of neurons identified were ON cells, 18% were OFF cells, and 32% Neutral cells. In sickle mice, 36% of neurons studied were ON cells, 23% were OFF cells, and 42% were Neutral cells. The proportions of cell types between control mice (n=66) and sickle mice (n=52) did not differ (chi-squared test; χ2=3.20, p=0.20). Since there was no significant difference in proportions of cell types between sickle and control mice, comparisons were made between male and female mice without regard to genotype. Of the 52 neurons studied in female mice (n=6), 52% were ON cells, 17% were OFF cells, and 31% were Neutral cells. Of the 66 neurons studied in male mice (n=10), 39% were ON cells, 21% were OFF cells, and 39% were Neutral cells. The distributions of cell types between male and female mice did not differ (chi-squared test; χ2=1.85, p=0.40).

Comparisons by Bonferroni t-test indicated that spontaneous activity (SA) of functional subtypes of neurons in the RVM did not differ between control and sickle mice. The mean SA of ON cells in control and sickle mice was 10 ± 3.0 and 13.3 ± 4.8 imp/sec, respectively (p = 0.54), and SA of OFF cells in HbAA control and HbSS sickle mice were 16.6 ± 3.4 and 29.8 ± 9.96 imp/sec, respectively (p = 0.26). SA of Neutral cells was 12.6 ± 2.6 imps/sec in control mice and 13.4 ± 7.0 imp/sec in sickle mice (p = 0.92).

Responses of RVM neurons to heat stimuli

To compare responsiveness of ON, OFF, or Neutral neurons in the RVM to heat stimuli between control and sickle mice we determined response thresholds, the number of heat-evoked action potentials, and the duration of evoked response.

ON cells.

In contrast to HbAA control mice, ON cells in sickle mice exhibited sensitization to heat. Figure 3A shows responses of representative ON cells to heat stimulation in sickle and control mice. The ON cells from sickle mice had a lower response threshold, and increased response with longer duration. Moreover, the EMG response in sickle mice appeared at a lower stimulus temperature of stimulation, indicating a decrease in threshold of the nociceptive withdrawal reflex. In sickle mice, the mean heat response threshold for ON cells (N=18) was lower (43.2 ± 0.71°C) as compared to those in control mice (N=35) (45.8 ± 0.49°C, p<0.01, Figure 3B).

Figure 3.

Figure 3.

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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.

Since EMG responses were always recorded in parallel with neuronal responses evoked by heat, we compared thresholds for evoking EMG in control and sickle mice. The mean heat threshold for evoking an EMG response in sickle mice was 45.6 ± 0.76 °C, which was lower than that for evoking an EMG response in control mice (47.7 ± 0.41 °C; p<0.05). Thus, the decrease in heat threshold for evoking EMG in sickle mice was consistent with the decrease in heat response threshold of ON cells.

The number of heat-evoked action potentials in those ON cells of sickle mice was more than 3 times greater (786.6 ± 208.7 impulses, N=18) compared to control mice (250.2 ± 44.1 impulses, p<0.005, N=35) (Figure 3C). Also, the duration of the response in ON cells in sickle mice (26.5 ± 6.0 sec) was more than 2 times greater than in control mice (11.8 ± 1.9 sec, p<0.01) (Figure 3D).

OFF cells.

In contrast to ON cells, responses of OFF cells to heat were not different between sickle and control mice (Figure 4A). Evoked responses of OFF cells in sickle and control mice were similar despite a decrease in threshold of the accompanying EMG response in sickle mice. The mean heat response threshold of OFF cells (N=11) in sickle mice was 45.6 ± 1.4 °C, and the response threshold of OFF cells (N=10) in control mice was 45.2 ± 0.79 °C (p=0.802, Figure 4B). Also, there were no differences in the decrease in the number of action potentials for OFF cells evoked by heat between sickle (−924.4 ± 472. 5 impulses) and control mice (−879.2 ± 295.3 impulses, p=0.938, Figure 4C). Similarly, the duration of the heat-evoked off response did not differ between the groups (52.2 ± 14.4 sec in sickle mice and 53.3 ± 15.1 sec in control mice, p=0.961, Figure 4D).

Figure 4.

Figure 4.

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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.

Neutral cells.

Neutral cells in sickle (n=23) and control (n=21) mice did not exhibit any responses to thermal stimulation of the paw (Figure 5); however, the accompanying EMG responses in sickle mice occurred at a lower threshold as compared to those in control mice. This was consistent with the threshold for EMG response while recording from ON and OFF cells.

Figure 5.

Figure 5.

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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.

Responses of RVM neurons to mechanical stimulation

ON cells.

Responses to mechanical stimuli were evoked using a calibrated von Frey monofilament (26 g); however, not all neurons responded to this stimulus. 10 of 15 (67%) ON cells in sickle mice and 12 of 17 (71%) ON cells in control mice exhibited a response. Only cells that were responsive to this mechanical stimulus were included in the analyses. Figure 6A and B show representative examples of responses to mechanical stimulation for an ON cell and the mean numbers of evoked impulses. The number of evoked action potentials for ON cells was more than 3 times greater in sickle mice (22.9 ± 3) than in control mice (6.6 ± 2.0, t-test, p<0.001).

Figure 6.

Figure 6.

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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.

OFF cells.

Similarly, not all OFF cells responded to the 26 g von Frey monofilament. In sickle mice, 5 of 7 (71%) responded whereas 4 of 6 (67%) OFF cells in control mice exhibited a response. As shown in Figure 6C and D, the number of evoked action potentials did not differ significantly between sickle (−16.7 ± 10.4) and control mice (−7.5 ± 2.0, t-test, p=0.461).

Neutral cells.

None of the 12 Neutral cells in sickle mice and the 9 Neutral cells in control mice exhibited any response to mechanical stimulation.

DISCUSSION

The RVM is part of a major descending pathway that modulates nociceptive transmission in the spinal cord and contributes to central sensitization and hyperalgesia. We (Pacharinsak et al., 2008) and others (Burgess et al., 2002; Chen et al., 2004; De Felice et al., 2011; Havelin et al., 2016; Kincaid et al., 2006; Nones et al., 2017; Pertovaara et al., 1996; Rivat et al., 2009; Saadé et al., 2012; Vanderah et al., 2001; Vera-Portocarrero et al., 2006) have used injection of local anesthetics into the RVM to determine the contribution of the RVM to the maintenance of hyperalgesia in various pain models. Similar to earlier studies, lidocaine into the RVM did not alter withdrawal responses in naïve C57B mice but abolished hyperalgesia in sickle mice. Thus, our results indicate that hyperalgesia in sickle mice is maintained, at least in part, by activation of descending pronociception from the RVM. In this regard, descending serotonergic pathways can facilitate or inhibit pain transmission (Dogrul et al., 2009; Ossipov et al., 2010; Suzuki et al., 2004). In earlier studies, we observed that serotonergic transmission from RVM produced antinociception in sickle mice (Tran et al., 2021). Treatment of sickle mice with duloxetine, a serotonin and norepinephrine reuptake inhibitor, decreased mechanical, thermal and deep tissue hyperalgesia. Complementary to these observations, treatment of sickle mice with p-chlorophenylalanine (PCPA), a rate-limiting enzyme in the biosynthesis of serotonin, also decreased hyperalgesia. These studies further suggest a role for the RVM in maintaining hyperalgesia in sickle mice. It should be noted that changes in many areas of the brain are likely to contribute to pain in SCD. For example, neuroimaging studies in patients with SCD examined functional resting state brain activity and functional connectivity and observed alterations in several brain regions that are involved in pain Case et al., 2017; Coloigner et al., 2017; Darbari et al., 2015). Interestingly, functional MRI studies in sickle mice showed that they had reduced BOLD responses following hypoxia (Wang et al., 2017). Structural changes that include a decrease in brain grey matter volume have also been observed in patients with SCD (Wang et al., 2021).

Although lidocaine into the RVM decreased hyperalgesia, descending modulation from the RVM is likely not the only mechanism that facilitates nociceptive transmission in the spinal cord of sickle mice. A variety of changes were found in the spinal cord that may contribute to increased excitability of spinal neurons, including elevated levels of substance P (SP), TLR4, COX-2, IL-6, phosphorylated mitogen-activated protein kinases (MAPKs), p44/p42 extracellular signal-regulated kinase (ERK), reactive oxygen species (ROS) (Cataldo et al., 2015; Lei et al., 2021) and other changes including activation of microglia and astrocytes (Lei et al., 2021; Valverde et al., 2016). Each of these changes may contribute to sensitization of dorsal horn neurons. Because many changes in the spinal cord can contribute to sensitization of dorsal horn neurons, the RVM likely plays a modulating, rather than a permissive, role in the development of sensitization of spinal neurons and hyperalgesia (Foo and Mason, 2003;Jinks et al., 2004; Khasabov et al., 2017; Khasabov and Simone, 2013). Also, sensitization of nociceptors to heat has been reported in sickle mice (Uhelski et al., 2017) and this is mediated, at least in part, through TRPV1 since sickle mice exhibited more nocifensive behaviors, and their DRG neurons were more responsive, to TRPV1 agonists (Sadler et al., 2018). Thus, we suggest that hyperalgesia in SCD is triggered at the spinal level by increased input from nociceptors and is maintained by descending facilitation from the RVM.

To determine changes in the RVM that may facilitate nociceptive transmission in the spinal cord and thereby contribute to hyperalgesia, we compared electrophysiological responses of RVM neurons between sickle and control mice. We found that ON cells, but not OFF cells, were sensitized to mechanical and heat stimuli in sickle mice. Responses of ON cells evoked by mechanical and heat stimuli were 3-fold greater in sickle mice as compared to control mice. Importantly, we reported earlier that sensitization of ON cells alone, produced by injection of substance P (SP) into the RVM (Budai et al., 2007), produced hyperalgesia and increased responses of nociceptive dorsal horn neurons (Khasabov et al., 2017). These studies were consistent with earlier studies showing that activation of NK-1 receptors in the RVM contributes to hyperalgesia (Hamity et al., 2010). In the present study, we did not determine if ON cells were sensitized to cold. Responses of RVM to cold should be investigated in the future since mice with SCD exhibit cold hyperalgesia (Sadler et al., 2018; Zappia et al., 2014) and little is known about underlying mechanisms.

A shift in the balance of pronociceptive output from the RVM may consist of an increase in ON cell activity and/or suppression of OFF cell activity. For example, in earlier studies with inflammation (Cleary and Heinricher, 2013), both ON cells and OFF cells exhibited time-dependent increases in responses where increases in responses in ON cells occurred early after inflammation and increased inhibitory responses of OFF cells occurred later. We also reported increased responses of ON cells following inflammation (Khasabov et al., 2012). An increase in pronociceptive output from the RVM was also suggested to contribute to cancer pain since descending serotonergic pathways facilitated pain and the 5HT-3 receptor antagonist, ondansetron, decreased cancer-evoked hyperalgesia (Donovan-Rodriguez et al., 2004). Interestingly responses of both ON and OFF cells were increased in a model of neuropathic pain (Carlson et al., 2007), and following intraplantar injection of capsaicin (Brink et al., 2012). In the present study, OFF cell activity was not changed in sickle mice, suggesting that sensitization of ON cells alone contributes to RVM-mediated facilitation of pain in sickle mice. SCD is associated with chronic inflammation, and the selective sensitization of ON cells in sickle mice is consistent with our earlier results showing sensitization of ON cells alone in a model of inflammatory pain produced by complete Freund’s adjuvant (Khasabov et al., 2012). Additional studies are needed to determine mechanisms underlying the sensitization of RVM neuronal subtypes as a function of the type of injury.

We also compared the proportions of cell types in the RVM of sickle and control mice because a change in the functional properties of cells could alter the balance of descending facilitation and inhibition without neuronal sensitization. No differences were found in the proportions of ON, OFF and Neutral cells between sickle and control mice. Our results are largely consistent with previous findings regarding cell proportions in models of inflammatory pain (Khasabov et al., 2015), indicating that the functional properties of neurons in the RVM did not change. We acknowledge that anesthesia could affect classification of RVM neurons. Deep anesthesia can reduce responses of ON and OFF cells, making them appear similar to Neutral cells (Leung and Mason, 1995). However, the proportion of Neutral cells did not differ between control and sickle mice. Furthermore, anesthesia may reduce EMG responses. In the present study all evoked neuronal responses were accompanied by a robust withdrawal response, decreasing the likelihood that anesthesia played a significant role in determining cell classification.

The mechanisms by which ON cells become sensitized in sickle mice are unknown, although several mediators that sensitize ON cells have been identified. For example, NK-1 receptors are located on ON cells in the RVM (Budai et al., 2007), and pathways from the PAG to the RVM contain SP (Nakaya et al., 1994). Injection of SP into the RVM sensitized ON, but not OFF, cells (Budai et al., 2007) and produced hyperalgesia. In addition, blockade of NK-1 receptors in the RVM decreased hyperalgesia produced by intraplantar injection of capsaicin (Khasabov et al., 2017; Pacharinsak et al., 2008). These studies show that activation and sensitization of ON cells by SP can produce hyperalgesia. Thus, the results of the present study showing functional changes only in the ON cells in the RVM in sickle mice align with previous work implicating NK-1 receptors in central sensitization. Importantly, neurons in the RVM may not only be influenced by descending sources such as the PAG, but also by ascending inputs from the spinal cord. An ascending pathway between the spinal cord and pain-modulating areas in the brain that contains NK-1 receptors was shown to contribute to hyperalgesia (Suzuki et al., 2002). Importantly, SP is increased in blood of patients with SCD (Brandow and Panepinto, 2016), and in the spinal cord of sickle mice (Wang et al., 2019), which might have a role in sensitizing ON cells and nociceptive neurons in the spinal cord that possess NK-1 receptors. These observations are consistent with amelioration of hyperalgesia in sickle mice with a combination of electroacupuncture and NK-1 receptor antagonism (Wang et al., 2019).

Cholecystokinin, brain-derived neurotrophic factor, tumor necrosis factor-ɑ, and serotonin, may also contribute to sensitization in the RVM (Gu et al., 2011; Guo et al., 2006; Heinricher and Neubert, 2004; Wei et al., 2008). An increase in NMDA receptors was observed in the brainstem following tissue inflammation (Miki et al., 2002), and it is possible that a similar mechanism of increased NMDA activity underlies sensitization in sickle mice. Studies exploring changes in cellular signaling including MAPK activity, CREB activity, and p38 (Bachstetter and van Eldik, 2010; Lonze and Ginty, 2002) would help elucidate the mechanistic basis for the changes observed in ON cells in the RVM of sickle mice.

In summary, our results show that the RVM maintains hyperalgesia in sickle mice through the sensitization of ON cells. The increased pronociceptive output suggests that the RVM contributes to the maintenance of sensitization of nociceptive neurons in the dorsal horn of the spinal cord, which in turn leads to hyperalgesia and chronic pain. It is unknown if changes in activity of RVM neurons contribute to the severe acute pain associated with vaso-occlusive crises that can result in hospitalization for SCD patients. Additional studies that elucidate the mechanisms by which ON cells are sensitized in sickle mice, and if the RVM contributes to acute pain in SCD, might lead to new targets within this descending pathway that may prove beneficial in managing acute and chronic pain in SCD.

Highlights.

  • Hyperalgesia in mice with SCD is mediated by sensitization of spinal neurons.

  • We determined if the RVM contributes to hyperalgesia in sickle mice.

  • Blockade of the RVM with lidocaine attenuated hyperalgesia in sickle mice.

  • Electrophysiology showed that ON cells in the RVM of sickle mice were sensitized.

  • Sensitization of ON cells in the RVM contributes to hyperalgesia in sickle mice.

Acknowledgements

This work was supported by NIH grants CA241627, HL135895 (DAS), CA263777 (SGK) and U18 EB029354, CA263806 and HL147562 (KG).

Conflict of interest statement

Kalpna Gupta: Honoraria: Novartis and CSL Behring. Research Grants: Cyclerion, 1910 Genetics, Novartis, Grifols, UCI Foundation and SCIRE Foundation. The other authors declare no competing financial interest.

Footnotes

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