Glutamatergic neurons and myeloid cells in the anterior cingulate cortex mediate secondary hyperalgesia in chronic joint inflammatory pain

Highlights

• Peripheral joint inflammation elicits secondary hyperalgesia in mice.

• Enhanced ACC glutamatergic neuronal activity in mice with secondary hyperalgesia.

• Neuron-myeloid cell interaction in the ACC is involved in secondary hyperalgesia.

Introduction

Joint inflammation, owing to local articular damage or immune mechanisms, is thought to be one of the common sources of arthritis-related pain (Alivernini et al., 2020, Culemann et al., 2019, Kraus et al., 2016). In response to joint inflammation-derived peripheral nociceptive input, chronic pain develops over time with centrally mediated neural sensitization. Secondary hyperalgesia, which occurs beyond the site of inflamed joints and affects multiple sites throughout the body (e.g., comorbid fibromyalgia symptoms), is widely believed to result from central sensitization (Neogi et al., 2016, Walsh and McWilliams, 2014). Once central sensitization occurs, arthritic patients poorly respond to commonly used oral analgesics and surgical procedures (Kolasinski et al., 2020, Geenen et al., 2018). For example, some patients with secondary hyperalgesia are at increased risk of developing chronic post-surgical pain despite receiving arthroplasty (Kurien et al., 2018, Wluka et al., 2020). Unfortunately, the neural mechanisms underlying secondary hyperalgesia remain largely unclear.

Joint inflammation-induced central sensitization is usually mediated by neuronal plasticity and myeloid cell activation at both spinal and supraspinal levels (Pan et al., 2021, Ghilardi et al., 2012). Neuroimaging studies have reported that arthritic patients with central sensitization exhibit functional and structural changes in multiple brain regions, such as the anterior cingulate cortex (ACC) (Reckziegel et al., 2016, Hassan and Walsh, 2014). Patients with painful hand osteoarthritis (OA) exhibit reduced ACC grey matter volume, which persists despite an improvement after pain treatment (Russell et al., 2018). Furthermore, patients with knee OA and concomitant central sensitization have altered functional connectivity networks involving the ACC, the latter of which is associated with a worse outcome following knee arthroplasty (Soni et al., 2019). Considering the point that ACC itself is a critical cortical area for central modulation of nociceptive information in inflammatory pain (Qiu et al., 2021, Chen et al., 2012), neuroimaging evidence suggests that ACC has high clinical relevance to central sensitization of arthritis and pain experience.

Myeloid cells in the central nervous system, such as microglia, dynamically survey and rapidly respond to any minor change in the spinal cord and brain (Nimmerjahn et al., 2005, Davalos et al., 2005). Many studies have shown that spinal myeloid cells are activated and involved in various models of arthritis, while microglial inhibitors can alleviate microgliosis and relieve the development of pain sensitivity (Tran et al., 2017, Fernandez-Zafra et al., 2019, Mousseau et al., 2018). However, less attention has been paid to the involvement of myeloid cell activation and the resultant inflammation in the supraspinal mechanism underlying pain processing in response to peripheral inflammation (Su et al., 2021). A previous study demonstrated an arthritis-induced myeloid response in several brain regions, including the cortex, striatum, and thalamus; however, the details regarding the histology and pain threshold changes are currently unclear (Süß et al., 2020). In addition, in mouse models of inflammatory pain, neuronal hyperexcitability, in terms of both pre- and post-synaptic long-term potentiation, has been reported in the ACC (Koga et al., 2015, Sellmeijer et al., 2018, Li et al., 2021). However, it remains unclear how dysfunction of the ACC, such as from myeloid cells and/or glutamatergic neurons may mediate joint inflammation-induced secondary hyperalgesia.

In this study, we established complete Freund’s adjuvant (CFA)-induced unilateral knee joint inflammation in mice and showed that it elicited secondary hypersensitivity in the ipsilateral hind-limb paw. Using a combination of immunofluorescence staining, three-dimensional reconstruction, flow cytometry, fiber photometry, real-time PCR analysis, electrophysiology, and chemogenetics, our cellular-level findings revealed that early joint inflammation contributed to secondary hypersensitivity at later time points in a neuroimmune interaction-dependent manner. Taken together, our findings offer a plausible and experimentally tractable framework for elucidating the cellular mechanisms by which ACC myeloid cells activate and induce hyperactivity of glutamatergic neurons to mediate joint inflammation-induced secondary hypersensitivity in mice.