Background & Aims

Sickle cell disease (SCD), the most common heritable hemoglobinopathy, affects nearly 8 million people worldwide. Individuals with SCD suffer from complex stimulus-evoked and ongoing pain, mediated in part by hyperexcitable peripheral sensory neurons. However, it is unclear whether acute or chronic elevation of cell-free heme, a key pathological feature of SCD, contributes to the aberrant activity of sensory neurons in patients or the Townes mouse model of SCD. Thus, determining whether heme drives pain behavior and enhanced excitability of dorsal root ganglia (DRG) primary sensory neurons could lead to the development of targeted pain therapies for SCD. The aim of these experiments is to determine: 1) whether heme is an acute mediator of ongoing or evoked pain, 2) whether heme-dependent signaling drives peripheral or central sensitization, and 3) the molecular identity of heme-responsive DRG and dorsal horn neurons.

Methods

In the current experiments, we apply heme directly to sensory axon terminals in the skin of outbred WT mice via intraplantar injection to study the role of heme in acute nociceptive pain. Our approach includes stimulus-evoked pain behaviors including von Frey up/down for mechanical hypersensitivity and the dry ice test for cold hyperalgesia, as well as ongoing pain behavior assays including conditioned place aversion, open field and home cage mobility assays, facial grimace analysis, and gradient-based thermal preference. We further make use of in vitro FURA 2 calcium imaging and current clamp electrophysiology of primary cultured DRG neurons from WT mice to determine whether heme enhances the excitability of DRG neurons. To determine the role of chronically elevated heme in SCD, we use intraperitoneal (i.p.) injections of haptoglobin in the humanized Townes SCD or Townes WT mice to reduce heme and perform the same behavioral and electrophysiological test in these animals.

Results

When heme is applied by intraplantar injection to outbred WT mice, we observe a rapid (<30 minute) onset mechanical and cold hypersensitivity that resolves within 24 hours. In addition, heme-injected mice display acutely increased paw licking behavior, altered thermal preference, and trending decreases in open field mobility without changes in metrics of anxiety. When heme is depletion in Townes SCD mice using haptoglobin, we observe alleviation of their steady-state mechanical and cold hypersensitivity and partially rescued ongoing pain measured by the murine grimace score at 24 hours post injection. When applied directly to cultured DRG sensory neurons from outbred WT mice, heme induces calcium flux across the neural membrane and pilot current clamp electrophysiology data suggests it may induce or enhance spontaneous action potential (AP) firing.

Conclusions

Based on our observations, heme is a mediator of acute nociceptive pain that causes hypersensitivity to mechanical and cold stimuli. Interruption of ongoing heme signaling in SCD mice also reduces several indices of steady-state pain. These behavioral changes are likely due to peripheral neuron activity as heme drives calcium influx in sensory neurons in culture and may result in enhanced AP generation. Current work includes determining the sensitized receptors and classes of sensory neurons that can respond to heme using patch clamp electrophysiology, qPCR, and immunofluorescence of heme-induced immediately early genes. in At the same time, we are currently determining whether intraplantar injection of heme induces acute central sensitization in the dorsal horn measured by receptive field size and windup in wide-dynamic range (WDR) neurons. Future directions include depleting heme in SCD mice to determine its role in nociceptor hyperexcitability and pain.

References

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Presenting Author

Samuel Zorn

Poster Authors

Samuel Zorn

BSc(Hons)

Medical College of Wisconsin

Lead Author

E-mail

Topics

  • Specific Pain Conditions/Pain in Specific Populations: Acute Pain and Nociceptive Pain