Cold exposure induces vaso-occlusion and pain in sickle mice that depend on complement activation.

Vaso-occlusive pain episodes (VOE) cause severe pain in patients with sickle cell disease (SCD). Vaso-occlusive events promote ischemia/reperfusion pathobiology that activates complement. We hypothesized that complement activation is linked to VOE. We used cold to induce VOE in the Townes sickle homozygous for hemoglobin S (HbSS) mouse model and complement inhibitors to determine whether anaphylatoxin C5a mediates VOE. We used a dorsal skinfold chamber to measure microvascular stasis (vaso-occlusion) and von Frey filaments applied to the plantar surface of the hind paw to assess mechanical hyperalgesia in HbSS and control Townes mice homozygous for hemoglobin A (HbAA) mice after cold exposure at 10°C/50°F for 1 hour. Cold exposure induced more vaso-occlusion in nonhyperalgesic HbSS mice (33%) than in HbAA mice (11%) or HbSS mice left at room temperature (1%). Cold exposure also produced mechanical hyperalgesia as measured by paw withdrawal threshold in HbSS mice compared with that in HbAA mice or HbSS mice left at room temperature. Vaso-occlusion and hyperalgesia were associated with an increase in complement activation fragments Bb and C5a in plasma of HbSS mice after cold exposure. This was accompanied by an increase in proinflammatory NF-κB activation and VCAM-1 and ICAM-1 expression in the liver. Pretreatment of nonhyperalgesic HbSS mice before cold exposure with anti-C5 or anti-C5aR monoclonal antibodies (mAbs) decreased vaso-occlusion, mechanical hyperalgesia, complement activation, and liver inflammatory markers compared with pretreatment with control mAb. Anti-C5 or -C5aR mAb infusion also abrogated mechanical hyperalgesia in HbSS mice with ongoing hyperalgesia at baseline. These findings suggest that C5a promotes vaso-occlusion, pain, and inflammation during VOE and may play a role in chronic pain.

Responses of neurons in the primary somatosensory cortex to itch- and pain-producing stimuli in rats.

Understanding of cortical encoding of itch is limited. Injection of pruritogens and algogens into the skin of the cheek produces distinct behaviors, making the rodent cheek a useful model for understanding mechanisms of itch and pain. We examined responses of neurons in the primary somatosensory cortex by application of mechanical stimuli (brush, pressure, and pinch) and stimulations with intradermal injections of pruritic and algesic chemical of receptive fields located on the skin of the cheek in urethane-anesthetized rats. Stimuli included chloroquine, serotonin, ß-alanine, histamine, capsaicin, and mustard oil. All 33 neurons studied were excited by noxious mechanical stimuli applied to the cheek. Based on mechanical stimulation most neurons were functionally classified as high threshold. Of 31 neurons tested for response to chemical stimuli, 84% were activated by one or more pruritogens/partial pruritogens. No cells were activated by all five substances. Histamine activated the greatest percentage of neurons and evoked the greatest mean discharge. Importantly, no cells were excited exclusively by pruritogens or partial pruritogens. The recording sites of all neurons that responded to chemical stimuli applied to the cheek were located in the dysgranular zone (DZ) and in deep laminae of the medial border of the vibrissal barrel fields (VBF). Therefore, neurons in the DZ/VBF of rats encode mechanical and chemical pruritogens and algogens. This cortical region appears to contain primarily nociceptive neurons as defined by responses to noxious pinching of the skin. Its role in encoding itch and pain from the cheek of the face needs further study.NEW & NOTEWORTHY Processing of information related to itch sensation at the level of cerebral cortex is not well understood. In this first single-unit electrophysiological study of pruriceptive cortical neurons, we show that neurons responsive to noxious and pruritic stimulation of the cheek of the face are concentrated in a small area of the dysgranular cortex, indicating that these neurons encode information related to itch and pain.

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

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.

The nAChR Chaperone TMEM35a (NACHO) Contributes to the Development of Hyperalgesia in Mice.

Pain is a major health problem, affecting over fifty million adults in the US alone, with significant economic cost in medical care and lost productivity. Despite evidence implicating nicotinic acetylcholine receptors (nAChRs) in pathological pain, their specific contribution to pain processing in the spinal cord remains unclear given their presence in both neuronal and non-neuronal cell types. Here we investigated if loss of neuronal-specific TMEM35a (NACHO), a novel chaperone for functional expression of the homomeric α7 and assembly of the heteromeric α3, α4, and α6-containing nAChRs, modulates pain in mice. Mice with tmem35a deletion exhibited thermal hyperalgesia and mechanical allodynia. Intrathecal administration of nicotine and the α7-specific agonist, PHA543613, produced analgesic responses to noxious heat and mechanical stimuli in tmem35a KO mice, respectively, suggesting residual expression of these receptors or off-target effects. Since NACHO is expressed only in neurons, these findings indicate that neuronal α7 nAChR in the spinal cord contributes to heat nociception. To further determine the molecular basis underlying the pain phenotype, we analyzed the spinal cord transcriptome. Compared to WT control, the spinal cord of tmem35a KO mice exhibited 72 differentially-expressed genes (DEGs). These DEGs were mapped onto functional gene networks using the knowledge-based database, Ingenuity Pathway Analysis, and suggests increased neuroinflammation as a potential contributing factor for the hyperalgesia in tmem35a KO mice. Collectively, these findings implicate a heightened inflammatory response in the absence of neuronal NACHO activity. Additional studies are needed to determine the precise mechanism by which NACHO in the spinal cord modulates pain.