Microglia and Inhibitory Circuitry in the Medullary Dorsal Horn: Laminar and Time-Dependent Changes in a Trigeminal Model of Neuropathic Pain

Craniofacial neuropathic pain affects millions of people worldwide and is often difficult to treat. Two key mechanisms underlying this condition are a loss of the negative control exerted by inhibitory interneurons and an early microglial reaction. Basic features of these mechanisms, however, are still poorly understood. Using the chronic constriction injury of the infraorbital nerve (CCI-IoN) model of neuropathic pain in mice, we have examined the changes in the expression of GAD, the synthetic enzyme of GABA, and GlyT2, the membrane transporter of glycine, as well as the microgliosis that occur at early (5 days) and late (21 days) stages post-CCI in the medullary and upper spinal dorsal horn. Our results show that CCI-IoN induces a down-regulation of GAD at both postinjury survival times, uniformly across the superficial laminae. The expression of GlyT2 showed a more discrete and heterogeneous reduction due to the basal presence in lamina III of ‘patches’ of higher expression, interspersed within a less immunoreactive ‘matrix’, which showed a more substantial reduction in the expression of GlyT2. These patches coincided with foci lacking any perceptible microglial reaction, which stood out against a more diffuse area of strong microgliosis. These findings may provide clues to better understand the neural mechanisms underlying allodynia in neuropathic pain syndromes.

Microglia and Inhibitory Circuitry in the Medullary Dorsal Horn: Laminar and Time-Dependent Changes in a Trigeminal Model of Neuropathic Pain

Craniofacial neuropathic pain affects millions of people worldwide and is often difficult to treat. Two key mechanisms underlying this condition are a loss of the negative control exerted by inhibitory interneurons and an early microglial reaction. Basic features of these mechanisms, however, are still poorly understood. Using the chronic constriction injury of the infraorbital nerve (CCI-IoN) model of neuropathic pain in mice, we have examined the changes in the expression of GAD, the synthetic enzyme of GABA, and GlyT2, the membrane transporter of glycine, as well as the microgliosis that occur at early (5 days) and late (21 days) stages post-CCI in the medullary and upper spinal dorsal horn. Our results show that CCI-IoN induces a down-regulation of GAD at both postinjury survival times, uniformly across the superficial laminae. The expression of GlyT2 showed a more discrete and heterogeneous reduction due to the basal presence in lamina III of ‘patches’ of higher expression, interspersed within a less immunoreactive ‘matrix’, which showed a more substantial reduction in the expression of GlyT2. These patches coincided with foci lacking any perceptible microglial reaction, which stood out against a more diffuse areas of strong microgliosis. These findings may provide clues to better understand the neural mechanisms underlying allodynia in neuropathic pain syndromes.

Modulation of mechanosensory vibrissal responses in the trigeminocervical complex by stimulation of the greater occipital nerve in a rat model of trigeminal neuropathic pain

Background: Stimulation of the occipital or trigeminal nerves has been successfully used to treat chronic refractory neurovascular headaches such as migraine or cluster headache, and painful neuropathies. Convergence of trigeminal and occipital sensory afferents in the ‘trigeminocervical complex’ (TCC) from cutaneous, muscular, dural, and visceral sources is a key mechanism for the input-induced central sensitization that may underlie the altered nociception. Both excitatory (glutamatergic) and inhibitory (GABAergic and glycinergic) mechanisms are involved in modulating nociception in the spinal and medullary dorsal horn neurons, but the mechanisms by which nerve stimulation effects occur are unclear. This study was aimed at investigating the acute effects of electrical stimulation of the greater occipital nerve (GON) on the responses of neurons in the TCC to the mechanical stimulation of the vibrissal pad.Methods: Adult male Wistar rats were used. Neuronal recordings were obtained in laminae II-IV in the TCC in control, sham and infraorbital chronic constriction injury (CCI-IoN) animals. The GON was isolated and electrically stimulated. Responses to the stimulation of vibrissae by brief air pulses were analyzed before and after GON stimulation. In order to understand the role of the neurotransmitters involved, specific receptor blockers of NMDA (AP-5), GABAA (bicuculline, Bic) and Glycine (strychnine, Str) were applied locally.Results: GON stimulation produced a facilitation of the response to light facial mechanical stimuli in controls, and an inhibition in CCI-IoN cases. AP-5 reduced responses to GON and vibrissal stimulation and blocked the facilitation of GON on vibrissal responses found in controls. The application of Bic or Str significantly reduced the facilitatory effect of GON stimulation on the response to vibrissal stimulation in controls. However, the opposite effect was found when GABAergic or Glycinergic transmission was prevented in CCI-IoN cases.Conclusions: GON stimulation modulates the responses of TCC neurons to light mechanical input from the face in opposite directions in controls and under CCI-IoN. This modulation is mediated by GABAergic and Glycinergic mechanisms. These results will help to elucidate the neural mechanisms underlying the effectiveness of nerve stimulation in controlling painful craniofacial disorders, and may be instrumental in identifying new therapeutic targets for their prevention and treatment.

Involvement of Interleukin-18-Mediated Microglia/Astrocyte Interaction in Experimental Models of Migraine

Background The exact molecular mechanisms of migraine were not fully understood. Emerging evidence indicated that inflammation had a significant role in pathophysiological mechanisms of migraine. The present study aimed to investigate the role of Interleukin-18-Mediated Microglia/Astrocyte Interaction in the development of hyperpathia or allodynia induced by migraine. Methods Experimental rat model of migraine was established by repetitive inflammatory soup (IS) dural infusions. The expression of Interleukin-18 (IL-18) and IL-18 receptor (IL-18R) was examined by quantitative real-time polymerase chain reaction or Western blot. The expression of activated microglia and astrocytes was examined by Western blot or immunofluorescence. The expression of nuclear factor-kappa B (NF-κB) was examined by Western blot. TAK-242 and anti-IL-18 antibody were used to inhibit the activation of toll-like receptor 4 (TLR4) and IL-18 pathway, respectively. Results IS dural infusions elicited microglial activation and astrocytic activation. Meanwhile, IS dural infusions induced the upregulation of IL-18 and IL-18R in microglia and astrocytes, respectively. Blocking the IL-18 signaling pathway attenuated nociceptive behavior. Meanwhile, blocking IL-18 signaling also suppressed astrocytic activation, as well as activation of NF-κB. And IL-18 dural infusions induced nociceptive behavior and glia activation. Conclusions IL-18, a product of microglial toll-like receptor 4 (TLR4) activation, acted on IL-18R expressed in astrocyte, and subsequently stimulated activation of NF-κB, leading to the astrocytic activation. Together, the present results suggest that IL-18-mediated microglia/astrocyte interactions in the medullary dorsal horn may participate in the development of hyperpathia or allodynia induced by migraine.

Involvement of Interleukin-18-Mediated Microglia/Astrocyte Interaction in Experimental Models of Migraine

Background: The exact molecular mechanisms of migraine were not fully understood. Emerging evidence indicated that inflammation had a significant role in pathophysiological mechanisms of migraine. The present study aimed to investigate the involvement of Interleukin-18-Mediated Microglia/Astrocyte Interaction in the development of hyperpathia or allodynia induced by migraine. Methods: Experimental rat model of migraine was established by repetitive inflammatory soup (IS) dural infusions. The expression of Interleukin-18 (IL-18) and IL-18 receptor (IL-18R) was examined by quantitative real-time polymerase chain reaction or Western blot. The expression of activated microglia and astrocytes was examined by Western blot or immunofluorescence. The expression of nuclear factor-kappa B (NF-κB) was examined by Western blot. TAK-242 and anti-IL-18 antibody were used to inhibit the activation of toll-like receptor 4 (TLR4) and IL-18 pathway, respectively. Results: IS dural infusions elicited microglial activation and astrocytic activation. Meanwhile, IS dural infusions induced the upregulation of IL-18 and IL-18R in microglia and astrocytes, respectively. Blocking the IL-18 signaling pathway attenuated nociceptive behavior. Meanwhile, blocking IL-18 signaling also suppressed astrocytic activation, as well as activation of NF-κB. And IL-18 dural infusions induced nociceptive behavior and glia activation. Conclusions: IL-18, a product of microglial toll-like receptor 4 (TLR4) activation, acted on IL-18R expressed in astrocyte, and subsequently stimulated activation of NF-κB, leading to the astrocytic activation. Together, the present results suggest that IL-18-mediated microglia/astrocyte interactions in the medullary dorsal horn may participate in the development of hyperpathia or allodynia induced by migraine.