Neuropathic pain is characterized by mechanical allodynia induced by low-threshold myelinated Aβ-fiber activation. The original gate theory of pain proposes that inhibitory interneurons in the lamina II of the spinal dorsal horn (DH) act as "gate control" units for preventing the interaction between innocuous and nociceptive signals. However, our understanding of the neuronal circuits underlying pain signaling and modulation in the spinal DH is incomplete. Using a rat model, we have shown that the convergence of glycinergic inhibitory and excitatory Aβ-fiber inputs onto PKCγ + neurons in the superficial DH forms a feed-forward inhibitory circuit that prevents Aβ input from activating the nociceptive pathway. This feed-forward inhibition was suppressed following peripheral nerve injury or glycine blockage, leading to inappropriate induction of action potential outputs in the nociceptive pathway by Aβ-fiber stimulation. Furthermore, spinal blockage of glycinergic synaptic transmission in vivo induced marked mechanical allodynia. Our findings identify a glycinergic feed-forward inhibitory circuit that functions as a gate control to separate the innocuous mechanoreceptive pathway and the nociceptive pathway in the spinal DH. Disruption of this glycinergic inhibitory circuit after peripheral nerve injury has the potential to elicit mechanical allodynia, a cardinal symptom of neuropathic pain.
Microglia and peripheral monocytes contribute to hypersensitivity in rodent models of neuropathic pain. However, the precise respective function of microglia and peripheral monocytes has not been investigated in these models. To address this question, here we combined transgenic mice and pharmacological tools to specifically and temporally control the depletion of microglia and monocytes in a mouse model of spinal nerve transection (SNT). We found that although microglia and monocytes are required during the initiation of mechanical allodynia or thermal hyperalgesia, these cells may not be as important for the maintenance of hypersensitivity. Moreover, we demonstrated that either resident microglia or peripheral monocytes are sufficient in gating neuropathic pain after SNT. We propose that resident microglia and peripheral monocytes act synergistically to initiate hypersensitivity and promote the transition from acute to chronic pain after peripheral nerve injury.
Summary Peripheral nerve injury causes neuropathic pain accompanied by remarkable microgliosis in the spinal cord dorsal horn. However, it is still debated whether infiltrated monocytes contribute to injury-induced expansion of the microglial population. Here we found that spinal microgliosis predominantly results from local proliferation of resident microglia but not from infiltrating monocytes after spinal nerve transection (SNT), using two genetic mouse models (CCR2RFP/+:CX3CR1GFP/+ and CX3CR1creER/+:R26tdTomato/+ mice) as well as specific staining of microglia and macrophages. Pharmacological inhibition of SNT-induced microglial proliferation correlated with attenuated neuropathic pain hypersensitivities. Microglial proliferation is partially controlled by purinergic and fractalkine signaling, as CX3CR1−/− and P2Y12−/− mice show reduced spinal microglial proliferation and neuropathic pain. These results suggest that local microglial proliferation is the sole source of spinal microgliosis, which represents a potential therapeutic target for neuropathic pain management.
Microglial cells are critical in the pathogenesis of neuropathic pain and several microglial receptors have been proposed to mediate this process. Of these receptors, the P2Y12 receptor is a unique purinergic receptor that is exclusively expressed by microglia in the central nervous system (CNS). In this study, we set forth to investigate the role of P2Y12 receptors in microglial electrophysiological and morphological (static and dynamic) activation during spinal nerve transection (SNT)-induced neuropathic pain in mice. First, we found that a genetic deficiency of the P2Y12 receptor (P2Y12−/− mice) ameliorated pain hypersensitivities during the initiation phase of neuropathic pain. Next, we characterized both the electrophysiological and morphological properties of microglia in the superficial spinal cord dorsal horn following SNT injury. We show dramatic alterations including a peak at 3 days post injury in microglial electrophysiology while high resolution two-photon imaging revealed significant changes of both static and dynamic microglial morphological properties by 7 days post injury. Finally, in P2Y12−/− mice, these electrophysiological and morphological changes were ameliorated suggesting roles for P2Y12 receptors in SNT-induced microglial activation. Our results therefore indicate that P2Y12 receptors regulate microglial electrophysiological as well as static and dynamic microglial properties after peripheral nerve injury, suggesting that the microglial P2Y12 receptor could be a potential therapeutic target for the treatment of neuropathic pain.
Extracellular calcium concentrations in the brain fluctuate during neuronal activities and may affect the behavior of brain cells. Microglia are highly dynamic immune cells of the brain. However, the effects of extracellular calcium concentrations on microglial dynamics have not been investigated. Here, we addressed this question in mouse brain slices and in vivo using two-photon microscopy. We serendipitously found that extracellular calcium reduction induced microglial processes to converge at distinct sites, a phenomenon we termed microglial process convergence (MPCs). Our studies revealed that MPCs target neuronal dendrites independent of neuronal action potential firing and is mediated by ATP release and microglial P2Y12 receptors. These results indicate that microglia monitor and interact with neurons during conditions of cerebral calcium reduction in the normal and diseased brain.
Background: Neuropathic pain after nerve injury is severe and intractable, and current drug and non-drug therapies offer very limited pain relief. Hyperbaric oxygen (HBO2) has been clinically used for protection of the nervous system after acute injury. We investigated whether HBO2 treatment could prevent and/or attenuate neuropathic pain in animals and in patients. Methods: Mechanical allodynia and thermal hyperalgesia and neurochemical alterations of neuropathic pain were analysed in male, adult, Sprague-Dawley rats with sciatic nerve injury. Clinical trials were conducted in patients with idiopathic trigeminal neuralgia. Results: Repetitive HBO2 treatment [a combination of pressure at 3 atmosphere absolute (ATA) and pure oxygen] greatly inhibited behavioural signs of neuropathic pain manifested as thermal hyperalgesia and mechanical allodynia. Such an HBO2 treatment also inhibited nerve injury-induced induction of c-Fos and activation of astrocytes and increased phosphorylation of NR2B receptor and the subsequent Ca 2+-dependent signals in rats. Neither high pressure (up to 3 ATA) nor pure oxygen alone resulted in analgesic effect. In clinical trials, one course of HBO2 therapy (10 consecutive days) produced a rapid-onset, dose-dependent and long-lasting analgesic effects evidenced by the decreased doses of carbamazepine required for keeping patient pain at a minimum and decreased scores of visual analogue scales, which was used for patient's self-evaluation. Conclusions: These findings support that HBO2 therapy is an effective approach for treating neuropathic pain in both animals and human beings and suggest that neural protection, anti-inflammation and inhibition of nerve injury-induced altered neural activity may contribute to the analgesic effect of HBO2 therapy.
Background The authors’ previous studies have found that spinal protein kinase C γ expressing neurons are involved in the feed-forward inhibitory circuit gating mechanical allodynia in the superficial dorsal horn. The authors hypothesize that nerve injury enhances the excitability of spinal protein kinase C γ expressing interneurons due to disinhibition of the feed-forward inhibitory circuit, and enables Aβ primary inputs to activate spinal protein kinase C γ expressing interneurons. Methods Prkcg-P2A-tdTomato mice were constructed using the clustered regularly interspaced short palindromic repeats and clustered regularly interspaced short palindromic repeats-associated nuclease 9 technology, and were used to analyze the electrophysiologic properties of spinal protein kinase C γ expressing neurons in both normal conditions and pathologic conditions induced by chronic constriction injury of the sciatic nerve. Patch-clamp whole cell recordings were used to identify the nature of the dynamic synaptic drive to protein kinase C γ expressing neurons. Results Aβ fiber stimulation evoked a biphasic synaptic response in 42% (31 of 73) of protein kinase C γ expressing neurons. The inhibitory components of the biphasic synaptic response were blocked by both strychnine and bicuculline in 57% (16 of 28) of neurons. Toll-like receptor 5 immunoreactive fibers made close contact with protein kinase C γ expressing neurons. After nerve injury, the percentage of neurons double-labeled for c-fos and Prkcg-P2A-tdTomato in animals walking on a rotarod was significantly higher than that in the nerve injury animals (4.1% vs. 9.9%, 22 of 539 vs. 54 of 548,P < 0.001). Aβ fiber stimulation evoked burst action potentials in 25.8% (8 of 31) of protein kinase C γ expressing neurons in control animals, while the proportion increased to 51.1% (23 of 45) in nerve injury animals (P = 0.027). Conclusions The Prkcg-P2A-tdTomato mice the authors constructed provide a useful tool for further analysis on how the spinal allodynia gate works. The current study indicated that nerve injury enhanced the excitability of spinal protein kinase C γ expressing interneurons due to disinhibition of the feed-forward inhibitory circuit, and enabled Aβ primary inputs to activate spinal protein kinase C γ expressing interneurons. Editor’s Perspective What We Already Know about This Topic What This Article Tells Us That Is New
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