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Inhibition of Mammalian Target of Rapamycin (mTOR) Signaling in the Insular Cortex Alleviates Neuropathic Pain After Peripheral Nerve Injury

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Inhibition of Mammalian Target of Rapamycin (mTOR) Signaling in the Insular Cortex Alleviates Neuropathic Pain After Peripheral Nerve Injury

Minjee Kwon et al. Front Mol Neurosci.

Abstract

Injury of peripheral nerves can trigger neuropathic pain, producing allodynia and hyperalgesia via peripheral and central sensitization. Recent studies have focused on the role of the insular cortex (IC) in neuropathic pain. Because the IC is thought to store pain-related memories, translational regulation in this structure may reveal novel targets for controlling chronic pain. Signaling via mammalian target of rapamycin (mTOR), which is known to control mRNA translation and influence synaptic plasticity, has been studied at the spinal level in neuropathic pain, but its role in the IC under these conditions remains elusive. Therefore, this study was conducted to determine the role of mTOR signaling in neuropathic pain and to assess the potential therapeutic effects of rapamycin, an inhibitor of mTORC1, in the IC of rats with neuropathic pain. Mechanical allodynia was assessed in adult male Sprague-Dawley rats after neuropathic surgery and following microinjections of rapamycin into the IC on postoperative days (PODs) 3 and 7. Optical recording was conducted to observe the neural responses of the IC to peripheral stimulation. Rapamycin reduced mechanical allodynia and downregulated the expression of postsynaptic density protein 95 (PSD95), decreased neural excitability in the IC, thereby inhibiting neuropathic pain-induced synaptic plasticity. These findings suggest that mTOR signaling in the IC may be a critical molecular mechanism modulating neuropathic pain.

Keywords: insular cortex; mTOR; neuropathic pain; rapamycin; synaptic plasticity.

Figures

Figure 1
Figure 1
Development of mechanical allodynia in nerve-injured (NP) and sham-injured (Sham) rats. After nerve injury, rats developed significant neuropathic pain on postoperative day 1 (POD1), POD3 and POD7 compared with the sham group. Data are presented as means ± SEM. **P < 0.01 vs. Sham.
Figure 2
Figure 2
c-Fos and phospho (p)-extracellular signal-regulated kinase (ERK) expression in NP and Sham rats. (A) Histological clarification of rostral insular cortex (IC) with rat atlas. Subdivisions of the IC were included in the black square box. c-Fos- and p-ERK-positive cells in the AIV and AID were analyzed. AIV, agranular insular cortex ventral; AID, agranular insular cortex dorsal; DI, dysgranular insular cortex; GI, granular insular cortex; S2, secondary somatosensory cortex; Den, dorsal endopiriform nucleus; VCI, ventral part of claustrum; DCI, dorsal part of claustrum. (B) Microphotographs of c-FOS and p-ERK in the rostral IC (AIV and AID areas). The arrows mean positive cells for c-FOS or p-ERK. (C) Quantification of c-Fos- and (D) p-ERK-positive cells on POD3. (E) Quantification of c-Fos- and (F) p-ERK-positive cells on POD7. Scale bars, 200 μm. Data are presented as means ± SEM. *P < 0.05, **P < 0.01 vs. Sham.
Figure 3
Figure 3
Phosphorylation of mammalian target of rapamycin (mTOR), p70 ribosomal S6 protein kinase (P70S6K) and 4E binding protein (4EBP), and expression of postsynaptic density protein 95 (PSD95) and NMDAR2B in the IC on POD3 after nerve injury. (A,B) p-mTOR increased in the NP group compared with the Sham group, but total mTOR levels were not significantly different on POD3. (C,D) p-P70S6K increased in the NP group compared with the Sham group, but total P70S6K levels were not different. (E,F) p-4EBP increased in the NP group compared with the Sham group, but total 4EBP levels were not different. (G,H) PSD95 and NMDAR2B levels increased significantly in the NP group compared with the Sham group. The intensity of the phospho-form band was normalized to that of the total form, and the total form bands were normalized to β-actin protein. Data are presented as means ± SEM. *P < 0.05, **P < 0.01 vs. Sham.
Figure 4
Figure 4
Phosphorylation of mTOR, P70S6K and 4EBP and expressions of PSD95 and NMDAR2B in the IC on POD7 after nerve injury. (A,B) p-mTOR increased in the NP group compared with the Sham group, but total mTOR levels were not significantly different on POD7. (C,D) p-P70S6K increased in the NP group compared with the Sham group, but total P70S6K levels were not different. (E,F) p-4EBP increased in the NP group compared with Sham group, but total 4EBP levels were not different. (G,H) PSD95 and NMDAR2B levels increased significantly in the NP group compared with the Sham group. The intensity of the phospho-form band was normalized to that of the total form, and the total form bands were normalized to β-actin protein. Data are presented as means ± SEM. *P < 0.05, **P < 0.01 vs. Sham.
Figure 5
Figure 5
Intracranial administration of rapamycin attenuates mechanical hypersensitivity. (A) Experimental schematic highlighting the time points of cannula implantation, nerve injury, microinjection and behavioral testing. (B) Microinjection site into the IC (left) and a representative photograph of a coronal section (right). Scale bar, 1 mm. (C) Changes in paw withdrawal thresholds to mechanical stimulation after microinjection of rapamycin (Rapa group, Rapa) or vehicle (Vehicle group, Vehicle) on POD3. The arrow indicates the time point of microinjection. Significant differences between the rapamycin and vehicle groups were observed between 0.5 h and 24 h after microinjection. (D) Changes in paw withdrawal thresholds to mechanical stimulation after microinjection of rapamycin or vehicle on POD7. The arrow indicates the time point of microinjection. Similarly, the rapamycin and vehicle groups were significantly different between 1 h and 24 h after microinjection. Data are presented as means ± SEM. *P < 0.05, **P < 0.01 vs. Vehicle.
Figure 6
Figure 6
Microinjection of rapamycin reduces the markers of nociceptive activation. (A) Experimental schematic highlighting the time points of cannula implantation, nerve injury, microinjection and perfusion. (B) Microphotographs of c-FOS and p-ERK in the rostral IC. The arrows mean positive cells for c-FOS or p-ERK. On POD3, (C) the numbers of c-Fos-positive and (D) p-ERK-positive cells decreased significantly in the Rapa group compared with in the Vehicle group. On POD7, (E) the numbers of c-Fos-positive and (F) p-ERK-positive cells decreased significantly in the Rapa group compared with in the Vehicle group. Data are presented as means ± SEM. *P < 0.05, **P < 0.01 vs. Vehicle.
Figure 7
Figure 7
Microinjection of rapamycin reversed the upregulation of the mTOR pathway and expressions of PSD95 and NMDAR2B on POD3 after nerve injury. (A,B) p-mTOR levels decreased in the Rapa group compared with in the Vehicle group, but total mTOR levels were not significantly different. (C,D) p-P70S6K levels decreased in the Rapa group compared with in the Vehicle group, but total P70S6K levels were not different. (E,F) p-4EBP levels decreased in the Rapa group compared with in the Vehicle group, but total 4EBP levels were not different. (G) PSD95 and (H) NMDAR2B levels decreased in the Rapa group compared with in the Vehicle group. Data are presented as means ± SEM. *P < 0.05, **P < 0.01 vs. Vehicle.
Figure 8
Figure 8
Microinjection of rapamycin reversed the upregulation of the mTOR pathway and expressions of PSD95 and NMDAR2B on POD7 after nerve injury. (A,B) p-mTOR levels decreased in the Rapa group compared with in the Vehicle group, but total mTOR levels were not significantly different. (C,D) p-P70S6K levels decreased in the Rapa group compared with in the Vehicle group, but total P70S6K levels were not different. (E,F) p-4EBP levels decreased in the Rapa group compared with in the Vehicle group, but total 4EBP levels were not different. (G) PSD95 and (H) NMDAR2B levels decreased significantly in the Rapa group compared with in the Vehicle group. The intensity of the phospho-form band was normalized to that of the total form, and the total form bands were normalized to β-actin protein. Data are presented as means ± SEM. *P < 0.05, **P < 0.01 vs. Vehicle.
Figure 9
Figure 9
Changes in optical signals before and after treatment with rapamycin and vehicle. (A) Optical signals before and after vehicle treatment. (B) Optical signals before and after rapamycin treatment. (C) Peak amplitudes before and after vehicle and rapamycin treatments following peripheral electrical stimulation with 5.0 mA intensity. (D) Activated areas before and after vehicle and rapamycin treatments following peripheral electrical stimulation with 5.0 mA intensity. The peak amplitudes and activated areas induced by peripheral electrical stimulation after rapamycin treatment were reduced compared with those before rapamycin treatment. However, they are not significantly different in both before and after vehicle treatment. *P < 0.05, **P < 0.01.

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