Animal Studies

Osteoarthritis (OA) is a degenerative joint disease that causes chronic disability among the elderly. Despite recent advances in symptomatic management of OA by pharmacological and surgical approaches, there remains a lack of optimal approaches to manage inflammation in the joints, which causes cartilage degradation and pain. In this study, we investigated the efficacy and underlying mechanisms of nicotine exposure in attenuating joint inflammation, cartilage degradation, and pain in a mouse model of OA. A mouse model of OA was induced by injection of monosodium iodoacetate into the knee joint. Cell culture models were also used to study the efficacy and underlying mechanisms of nicotine treatment in attenuating symptoms of OA. Nicotine treatment reduced mechanical allodynia, cartilage degradation, and the upregulation of matrix metalloproteinase-9 (MMP-9), a hallmark of joint inflammation in OA, in mice treated with monosodium iodoacetate. The effects of nicotine were abolished by the selective α7 nicotinic acetylcholine receptor (nAChR) blocker, methyllycaconitine. In RAW264.7 cells and murine primary bone marrow-derived macrophages, nicotine significantly inhibited MMP-9 production induced by LPS. In addition, nicotine significantly enhanced PI3K/Akt and inhibited NF-κB translocation from the cytosol to the nucleus in an α7-nAChR-dependent manner, suggesting that nicotine acts on α7-nAChRs to inhibit MMP-9 production by macrophages through modulation of the PI3K/Akt-NF-κB pathway. Our results provide novel evidence that nicotine can attenuate joint inflammation and pain in experimental OA via α7-nAChRs. α7-nAChR could thus serve as a highly promising target to manage joint inflammation and pain in OA.

The dorsal root ganglia (DRG) contain the somas of first-order sensory neurons critical for somatosensation. Due to technical difficulties, DRG neuronal activity in awake behaving animals remains unknown. Here, we develop a method for imaging DRG at cellular and subcellular resolution over weeks in awake mice. The method involves the installation of an intervertebral fusion mount to reduce spinal movement, and the implantation of a vertebral glass window without interfering animals' motor and sensory functions. In vivo two-photon calcium imaging shows that DRG neuronal activity is higher in awake than anesthetized animals. Immediately after plantar formalin injection, DRG neuronal activity increases substantially and this activity upsurge correlates with animals' phasic pain behavior. Repeated imaging of DRG over 5 weeks after formalin injection reveals persistent neuronal hyperactivity associated with ongoing pain. The method described here provides an important means for in vivo studies of DRG functions in sensory perception and disorders.

Chronic pain presents a major unmet clinical problem. The development of more effective treatments is hindered by our limited understanding of the neuronal circuits underlying sensory perception. Here, we show that parvalbumin (PV)-expressing dorsal horn interneurons modulate the passage of sensory information conveyed by low-threshold mechanoreceptors (LTMRs) directly via presynaptic inhibition and also gate the polysynaptic relay of LTMR input to pain circuits by inhibiting lamina II excitatory interneurons whose axons project into lamina I. We show changes in the functional properties of these PV interneurons following peripheral nerve injury and that silencing these cells unmasks a circuit that allows innocuous touch inputs to activate pain circuits by increasing network activity in laminae I-IV. Such changes are likely to result in the development of tactile allodynia and could be targeted for more effective treatment of mechanical pain.

Piezo channels are mechanically activated ion channels that confer mechanosensitivity to a variety of different cell types. Piezos oligomerize as propeller-shaped homotrimers that are thought to locally curve the membrane into spherical domes that project into the cell. While several studies have identified domains and amino acids that control important properties such as ion permeability and selectivity as well as inactivation kinetics and voltage sensitivity, only little is known about intraprotein interactions that govern mechanosensitivity-the most unique feature of PIEZOs. Here we used site-directed mutagenesis and patch-clamp recordings to investigate the mechanogating mechanism of PIEZO2. We demonstrate that charged amino acids at the interface between the beam domain-i.e., a long α-helix that protrudes from the intracellular side of the "propeller" blade toward the inner vestibule of the channel-and the C-terminal domain (CTD) as well as hydrophobic interactions between the highly conserved Y2807 of the CTD and pore-lining helices are required to ensure normal mechanosensitivity of PIEZO2. Moreover, single-channel recordings indicate that a previously unrecognized intrinsically disordered domain located adjacent to the beam acts as a cytosolic plug that limits ion permeation possibly by clogging the inner vestibule of both PIEZO1 and PIEZO2. Thus, we have identified several intraprotein domain interfaces that control the mechanical activation of PIEZO1 and PIEZO2 and which might thus serve as promising targets for drugs that modulate the mechanosensitivity of Piezo channels.

We explored the immune neuropathology underlying multi-day relief from neuropathic pain in a rat model initiated at the sciatic nerve, by using a nanoemulsion-based nanomedicine as a biological probe. The nanomedicine is theranostic: both therapeutic (containing celecoxib drug) and diagnostic (containing near-infrared fluorescent (NIRF) dye) and is small enough to be phagocytosed by circulating monocytes. We show that pain-like behavior reaches a plateau of maximum hypersensitivity 8 days post-surgery, and is the rationale for intravenous delivery at this time-point. Pain relief is evident within 24 h, lasting approximately 6 days. The ipsilateral sciatic nerve and associated L4 and L5 dorsal root ganglia (DRG) tissue of both nanomedicine and control (nanoemulsion without drug) treated animals was investigated by immunofluorescence and confocal microscopy at the peak of pain relief (day-12 post-surgery), and when pain-like hypersensitivity returns (day-18 post-surgery). At day-12, a significant reduction of infiltrating macrophages, mast cells and mast cell degranulation was observed at the sciatic nerve following treatment. In the DRG, there was no effect of treatment at both day-12 and day-18. Conversely, at the DRG, there is a significant increase in macrophage infiltration and mast cell degranulation at day-18. The treatment effect on immune pathology in the sciatic nerve was investigated further by assessing the expression of macrophage cyclooxygenase-2 (COX-2)-the drug target-and extracellular prostaglandin E2 (PGE2), as well as the proportion of M1 (pro-inflammatory) and M2 (anti-inflammatory) macrophages. At day-12, there is a significant reduction of COX-2 positive macrophages, extracellular PGE2, and a striking reversal of macrophage polarity. At day-18, these measures revert to levels observed in control-treated animals. Here we present a new paradigm of immune neuropathology research, by employing a nanomedicine to target a mechanism of neuropathic pain-resulting in long-lasting pain relief–whilst revealing novel immune pathology at the injured nerve and associated DRG.