Peripheral macrophages contribute to nociceptor priming in mice with chronic intermittent hypoxia.

Obstructive sleep apnea (OSA) is a prevalent sleep disorder that is associated with increased incidence of chronic musculoskeletal pain. We investigated the mechanism of this association in a mouse model of chronic intermittent hypoxia (CIH) that mimics the repetitive hypoxemias of OSA. After 14 days of CIH, both male and female mice exhibited behaviors indicative of persistent pain, with biochemical markers in the spinal cord dorsal horn and sensory neurons of the dorsal root ganglia consistent with hyperalgesic priming. CIH, but not sleep fragmentation alone, induced an increase in macrophage recruitment to peripheral sensory tissues (sciatic nerve and dorsal root ganglia), an increase in inflammatory cytokines in the circulation, and nociceptor sensitization. Peripheral macrophage ablation blocked CIH-induced hyperalgesic priming. The findings suggest that correcting the hypoxia or targeting macrophage signaling might suppress persistent pain in patients with OSA.

PACAP38/mast-cell-specific receptor axis mediates repetitive stress-induced headache in mice.

BACKGROUND: Pain, an evolutionarily conserved warning system, lets us recognize threats and motivates us to adapt to those threats. Headache pain from migraine affects approximately 15% of the global population. However, the identity of any putative threat that migraine or headache warns us to avoid is unknown because migraine pathogenesis is poorly understood. Here, we show that a stress-induced increase in pituitary adenylate cyclase-activating polypeptide-38 (PACAP38), known as an initiator of allosteric load inducing unbalanced homeostasis, causes headache-like behaviour in male mice via mas-related G protein-coupled receptor B2 (MrgprB2) in mast cells. METHODS: The repetitive stress model and dural injection of PACAP38 were performed to induce headache behaviours. We assessed headache behaviours using the facial von Frey test and the grimace scale in wild-type and MrgprB2-deficient mice. We further examined the activities of trigeminal ganglion neurons using in vivo Pirt-GCaMP Ca2+ imaging of intact trigeminal ganglion (TG). RESULTS: Repetitive stress and dural injection of PACAP38 induced MrgprB2-dependent headache behaviours. Blood levels of PACAP38 were increased after repetitive stress. PACAP38/MrgprB2-induced mast cell degranulation sensitizes the trigeminovascular system in dura mater. Moreover, using in vivo intact TG Pirt-GCaMP Ca2+ imaging, we show that stress or/and elevation of PACAP38 sensitized the TG neurons via MrgprB2. MrgprB2-deficient mice showed no sensitization of TG neurons or mast cell activation. We found that repetitive stress and dural injection of PACAP38 induced headache behaviour through TNF-a and TRPV1 pathways. CONCLUSIONS: Our findings highlight the PACAP38-MrgprB2 pathway as a new target for the treatment of stress-related migraine headache. Furthermore, our results pertaining to stress interoception via the MrgprB2/PACAP38 axis suggests that migraine headache warns us of stress-induced homeostatic imbalance.

In Vivo Calcium Imaging Visualizes Incision-Induced Primary Afferent Sensitization and Its Amelioration by Capsaicin Pretreatment.

Previous studies have shown that infiltration of capsaicin into the surgical site can prevent incision-induced spontaneous pain like behaviors and heat hyperalgesia. In the present study, we aimed to monitor primary sensory neuron Ca2+ activity in the intact dorsal root ganglia (DRG) using Pirt-GCaMP3 male and female mice pretreated with capsaicin or vehicle before the plantar incision. Intraplantar injection of capsaicin (0.05%) significantly attenuated spontaneous pain, mechanical, and heat hypersensitivity after plantar incision. The Ca2+ response in in vivo DRG and in in situ spinal cord was significantly enhanced in the ipsilateral side compared with contralateral side or naive control. Primary sensory nerve fiber length was significantly decreased in the incision skin area in capsaicin-pretreated animals detected by immunohistochemistry and placental alkaline phosphatase (PLAP) staining. Thus, capsaicin pretreatment attenuates incisional pain by suppressing Ca2+ response because of degeneration of primary sensory nerve fibers in the skin.SIGNIFICANCE STATEMENT Postoperative surgery pain is a major health and economic problem worldwide with ∼235 million major surgical procedures annually. Approximately 50% of these patients report uncontrolled or poorly controlled postoperative pain. However, mechanistic studies of postoperative surgery pain in primary sensory neurons have been limited to in vitro models or small numbers of neurons. Using an innovative, distinctive, and interdisciplinary in vivo populational dorsal root ganglia (DRG) imaging (>1800 neurons/DRG) approach, we revealed increased DRG neuronal Ca2+ activity from postoperative pain mouse model. This indicates widespread DRG primary sensory neuron plasticity. Increased neuronal Ca2+ activity occurs among various sizes of neurons but mostly in small-diameter and medium-diameter nociceptors. Capsaicin pretreatment as a therapeutic option significantly attenuates Ca2+ activity and postoperative pain.

Visualization of Peripheral Neuron Sensitization in a Surgical Mouse Model of Osteoarthritis by In Vivo Calcium Imaging.

OBJECTIVE: To develop a method for analyzing sensory neuron responses to mechanical stimuli in vivo, and to evaluate whether these neuronal responses change after destabilization of the medial meniscus (DMM). METHODS: DMM or sham surgery was performed in 10-week-old male C57BL/6 wild-type or Pirt-GCaMP3+/- mice. All experiments were performed 8 weeks after surgery. Knee and hind paw hyperalgesia were assessed in wild-type mice. The retrograde label DiI was injected into the ipsilateral knee to quantify the number of knee-innervating neurons in the L4 dorsal root ganglion (DRG) in wild-type mice. In vivo calcium imaging was performed on the ipsilateral L4 DRG of Pirt-GCaMP3+/- mice as mechanical stimuli (paw pinch, knee pinch, or knee twist) were applied to the ipsilateral hind limb. RESULTS: Eight weeks after surgery, mice subjected to DMM had more hyperalgesia in the knee and hind paw compared to mice subjected to sham surgery. Intraarticular injection of DiI labeled similar numbers of neurons in the L4 DRG of mice subjected to sham surgery and mice subjected to DMM. Increased numbers of sensory neurons responded to all 3 mechanical stimuli in mice subjected to DMM, as assessed by in vivo calcium imaging. The majority of responses in mice subjected to sham surgery and mice subjected to DMM were in small to medium-sized neurons, consistent with the size of nociceptors. The magnitude of responses was similar between mice subjected to sham surgery and mice subjected to DMM. CONCLUSION: Our findings indicate that increased numbers of small to medium-sized DRG neurons respond to mechanical stimuli 8 weeks after DMM surgery, suggesting that nociceptors have become sensitized by lowering the response threshold.

Selective keratinocyte stimulation is sufficient to evoke nociception in mice.

The skin epidermis is densely innervated by peripheral sensory nerve endings. Nociceptive neurons, whose terminals are in close contact with epidermal keratinocytes, can be activated directly by noxious physical and chemical stimuli to trigger pain. However, whether keratinocytes can signal acutely to sensory nerve terminals to initiate pain in vivo remains unclear. Here, using the keratin 5 promoter to selectively express the capsaicin receptor TRPV1 in keratinocytes of TRPV1-knockout mice, we achieved specific stimulation of keratinocytes with capsaicin. Using this approach, we found that keratinocyte stimulation was sufficient to induce strong expression of the neuronal activation marker, c-fos, in laminae I and II of the ipsilateral spinal cord dorsal horn and to evoke acute paw-licking nocifensive behavior and conditioned place aversion. These data provide direct evidence that keratinocyte stimulation is sufficient to evoke acute nociception-related responses.

Streptococcus mutans GS-5 antigen I/II stimulates cell survival in serum deprived-cultures through PI3K/Akt pathways.

The antigen I/II (AgI/II) protein is a major surface protein that mediates the attachment of Streptococcus mutans (S. mutans) to the saliva-coated pellicle. Numerous studies have investigated not only the mechanisms by which AgI/II signaling is transduced within cells, but have also attempted to use AgI/II-specific antibodies to treat dental caries and host immune responses. However, little information is available about the effects of AgI/II on basic cellular events in bone cells. In this study, we examined the effects of the His-tagged recombinant N-terminal half of the AgI/II protein (rAgI/II-N) generated from S. mutans GS-5 on the viability, proliferation, and cell cycle progression of primary calvarial osteoblasts. We also investigated the mechanisms involved in the rAgI/II-N-mediated survival of serum-starved osteoblasts. We found that rAgI/II treatment attenuated the serum deprivation-induced decrease in cell viability and proliferation of osteoblasts. rAgI/II-N also prevented the loss of mitochondrial membrane potential (MMP), alterations in levels of two key mitochondrial Bcl-2 family proteins, and the accumulation of numerous cells into the sub-G(1) phase that were observed in serum-starved osteoblasts. Pharmacological inhibitors of phosphoinositide 3-kinase (PI3K), but not of extracellular signal-regulated kinase or Ras, blocked the rAgI/II-N-mediated protection against serum deprivation-induced cell death. Additional experiments revealed that the integrin α5ß1-mediated PI3K pathway is required for rAgI/II-N-mediated Akt phosphorylation in osteoblasts. Collectively, these results suggest that rAgI/II-N induces survival signals in serum-starved osteoblasts through integrin-induced PI3K/Akt signaling pathways.

Effects of gold nanorod concentration on the depth-related temperature increase during hyperthermic ablation.

The photothermal properties of gold nanorods (GNRs) provide an opportunity for the clinical application of highly efficient and tumor-specific photothermal therapy. For the effective hyperthermic ablation of tumor tissue using GNRs, it is essential to maintain a homogeneous therapeutic temperature in the target tissue during treatment. This study investigates whether the concentration of GNRs affects the distribution of the temperature increase during hyperthermal therapy. The investigation is conducted using polyacrylamide phantoms containing varying amounts of GNRs. In 0.1, 0.25, and 0.5 nM GNR-suspended phantoms, the change in temperature is relatively uniform along the depth of each phantom during laser irradiation at 2 W cm(-2) . In 1.0, 2.0, and 5.0 nM GNR-suspended phantoms, the rates of temperature increase in the deep regions of the phantoms decrease with increasing GNR concentration. At a laser irradiation of 5 W cm(-2) , the temperature of the GNR-suspended phantoms increases at a faster rate, whereas the range of GNR concentrations for maintaining the homogeneity of the temperature increase is not affected. This suggests that the concentration of GNRs is the major determinant of the depth-related temperature increase during hyperthermic ablation. Therefore, prior to the clinical application of hyperthermic ablation using GNRs, the concentration of GNRs has to be optimized to ensure a homogeneous distribution of therapeutic temperature in the targeted tissue.