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Male Sprague-Dawley rats weighing between and g were used in this study. All experiments conformed to the Australian National Health and Medical Research Council code of practice for the use of animals in research and were approved by the University of Melbourne Animal Experimentation Ethics Committee. Selective antagonists or function blocking antibodies that directly target Piezo2 are not yet available, and we are currently unable to make recordings from bone afferent neurons in mice so cannot take advantage of transgenic approaches to manipulation of Piezo2.
Instead, we used Piezo2 knockdown with antisense oligodeoxynucleotides ODNs to explore roles for Piezo2 in regulating the mechanical sensitivity of bone afferent neurons in rats Figure 1A. The approach of using antisense ODNs to attenuate the expression of proteins in peripheral nociceptors is well supported in the literature Khasar et al. We did not observe any differences in the function of bone afferent neurons between the two doses, so we report data from the two doses together as a single group.
Piezo2 knockdown approach and confirmation of protein knockdown. A Piezo2 antisense or mismatch oligodeoxynucleotides ODNs were administered by intrathecal injection. Intrathecal injections of the ODNs were made into the lumbar subarachnoid space with a Hamilton syringe, between L4 and L5 vertebra, once per day for three days before we performed electrophysiological experiments. Correct placement of the intrathecal injection was confirmed by the presence of a sudden tail flick or rapid limb flinch, both reflexes that occur in response to mechanical stimulation to the lumbar subarachnoid space.
Recovery was monitored continuously until the animal was conscious and freely moving without any noticeable disturbances usually after a few minutes. A fine branch of the tibial nerve that innervates the marrow cavity of the rat tibia was dissected and placed over a platinum hook electrode for electrophysiological recordings.
Mechanical stimulation of the axon terminals of bone afferent neurons innervating the marrow cavity was achieved by injecting heparinized physiological saline 0. Changes in intraosseous pressure were measured using a bridge-amplified TAM-D Amplifier, Harvard Apparatus signal derived from a pressure transducer APT Transducer, Harvard Apparatus placed to measure the input pressure to the bone.
The pump uses this as feedback to adjust flow through the system to control and maintain constant input pressures. These data were stored to PC in parallel with the nerve recordings. We were unable to routinely record conduction velocities in each experiment because we could not electrically stimulate the receptive fields of individual units buried deep inside the marrow cavity.
For a thorough discussion of how this division was selected, and to view the data that supports this classification, see Nencini et al.
We excluded units from analysis if their amplitude and duration changed over the course of the recording. We were not able to isolate individual C bone afferent neurons using our recording configuration so do not report on C fiber bone afferent neurons in this study. Discharge frequency was reported over the entire ramp-and-hold pressure stimulus total discharge frequency , for the ramp phase of the stimulus defined as the first 3 s of the ramp-and-hold pressure stimulus , and for the hold phase of the stimulus defined as the 5 s period starting 5 s after the beginning of the hold phase of the pressure stimulus.
Threshold for activation was assessed on the rising phase of the pressure ramp. To assess the conditioning effect of the ISIs, the threshold for activation and total discharge frequency at the second stimulus was presented as a percentage of that at the first baseline stimulus.
The concentration of NGF used is in the range that produces behavioral pain-like responses when applied to the footpad Mills et al. This 30 min ISI was used to avoid the effect of stimulus-evoked fatigue on discharge frequency observed with 15 min ISIs in Piezo2 knockdown animals. The thresholds for mechanical activation and the discharge frequency during the pressure stimulus were determined following injection of NGF and expressed as a percentage of pre-NGF injection baseline values.
The number of NGF sensitized vs. These data were re-analyzed for the purpose of the present study using the approaches outlined above. Results were analyzed using computer-assisted densitometry ImageLab Software v6. The density of each band was expressed as arbitrary units. A skin incision was made on the medial aspect of the tibia and a small hole was made in the cortical bone on the medial aspect of the tibial diaphysis using a sterile needle.
The hole was sealed with bone wax to prevent leakage into surrounding tissues. The area was washed extensively with 0. Skin incisions were closed. Animals were left for a day survival period to allow for transport of the tracer to neuronal cell bodies in the DRG.
Multiple series of sections were collected on gelatinized glass slides 0. The concentration of each of the primary antibodies was optimized in preliminary experiments. All antisera were diluted in PBS containing 0. Details of the primary and secondary antibodies used are provided in Table 1. To avoid double counting, and to sample from the widest part of the cell, only cells with a nucleus visible under the microscope were examined.
The counts presented are estimates of the total number of bone afferent neurons and may overestimate afferents with large cell bodies. We determined the proportion of retrograde labeled neurons that expressed each antibody marker for each animal. Individual images were contrast and brightness adjusted. No other manipulations were made to the images.
The ability of this antibody to detect Piezo2 has been confirmed by antibody-mediated affinity purification of native Piezo2 from mouse DRG, followed by mass spectrometry and label-free quantification Narayanan et al. Piezo2 was detected in samples treated with the Piezo2 antibody, but not in samples treated with its isotype control antibody Narayanan et al.
Western blot analysis reveals the antibody labels an 80 kDa band in mice, and that the intensity of the band is significantly reduced by siRNA treatment targeted specifically at Piezo2 Du et al. The 80 kDa band has also been reported using a different antibody in human vascular endothelial cells and rat bladder tissue, and siRNA mediated knockdown of this band is associated with reduced calcium signaling in human vascular endothelial cells Yang et al.
Mixed model analysis revealed significantly reduced discharge frequency calculated over the entire duration of the ramp-and-hold pressure stimulus total discharge frequency , in animals administered Piezo2 antisense ODNs relative to those administered mismatch ODNs [ F 3.
To determine if the reduced activity was confined to either the ramp phase when the pressure was changing, or the hold phase of the stimulus when pressure was constant, we repeated the analyses using data that were confined to each of these distinct parts of the ramp-and-hold pressure stimulus we applied. Mixed model analyses revealed a significant reduction in discharge frequency during both the ramp [F 3.
The reduction in discharge frequency was observed during both the D ramp [F 3. We have previously reported that repetitive mechanical stimulation can lead to significant changes in the response properties of bone afferent neurons Nencini and Ivanusic, Piezo2 knockdown prolongs stimulus evoked fatigue. A Schematic representation of the repetitive stimulation experimental protocol. The response to repetitive stimulation was measured at 15 and 30 min interstimulus intervals ISIs and compared between Piezo2 antisense and mismatch control treated animals.
We used retrograde tracing and immunohistochemistry to determine if Piezo2 expressing bone afferent neurons co-express the NGF receptor TrkA Figure 4A. A total of retrograde labeled bone afferent neurons were counted in L3 DRG taken from three animals.
These findings show that a substantial proportion of bone afferent neurons express both Piezo2 and the NGF receptor TrkA, and provide the necessary substrate for an interaction between Piezo2 and NGF to contribute to pain signaling. A Retrograde labeling of bone afferent neurons FB, blue, arrowheads revealed that a substantial proportion of neurons that innervate bone express Piezo2 red, hashes and the NGF receptor TrkA green, asterisks.
B Frequency histogram showing that the majority of medium-sized bone afferent neurons expressed Piezo2, and that most Piezo2 expressing bone afferent neurons also expressed TrkA. Destruction of bone by osteolytic processes or trauma can lead to mechanical injury or distortion of bone that activates mechanically sensitive nerve terminals in bone marrow to produce pain Haegerstam, ; Bove et al. Agents that are known to inhibit inflammatory processes reduce mechanically induced pain in animal models of bone cancer Honore and Mantyh, and pro-inflammatory cytokines contribute to mechanically induced pain in bone fracture models Li et al.
These studies highlight mechanical disturbances of the bone marrow as important components of bone pathology, and suggest that treatment strategies targeted specifically at mechanically induced pain will provide therapeutic benefit.
Bone pain is transmitted by two main classes of peripheral nociceptors Nencini and Ivanusic, C nociceptors are unmyelinated sensory neurons that encode slow, aching pain relevant to more chronic conditions such as osteoarthritis or bone cancer.
Phasic-tonic units respond best to the intensity of sustained intra-osseous pressure and may signal pain associated with pathologies that involve sustained increases in pressure within bone, for example intra-osseous engorgement syndrome. In contrast, phasic units respond best to the rate of change in intra-osseous pressure and are likely to signal pain associated with rapid changes in pressure within the marrow cavity, for example during needle aspiration of bone marrow or emergency intra-osseous vascular access.
The response of single bone afferent neurons with C fiber conduction velocities has not yet been reported, but there is clear evidence that C bone afferent neurons in whole-nerve recordings can be activated by noxious mechanical stimuli applied to bone marrow Nencini et al.
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