Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 288 (33), 24116-27

Role of lipocalin-2-chemokine Axis in the Development of Neuropathic Pain Following Peripheral Nerve Injury

Affiliations

Role of lipocalin-2-chemokine Axis in the Development of Neuropathic Pain Following Peripheral Nerve Injury

Sangmin Jeon et al. J Biol Chem.

Abstract

Lipocalin 2 (LCN2), which is also known as 24p3 and neutrophil gelatinase-associated lipocalin (NGAL), binds small, hydrophobic ligands and interacts with cell surface receptor 24p3R to regulate diverse cellular processes. In the present study, we examined the role of LCN2 in the pathogenesis of neuropathic pain using a mouse model of spared nerve injury (SNI). Lcn2 mRNA levels were significantly increased in the dorsal horn of the spinal cord after SNI, and LCN2 protein was mainly localized in neurons of the dorsal and ventral horns. LCN2 receptor 24p3R was expressed in spinal neurons and microglia after SNI. Lcn2-deficient mice exhibited significantly less mechanical pain hypersensitivity during the early phase after SNI, and an intrathecal injection of recombinant LCN2 protein elicited mechanical pain hypersensitivity in naive animals. Lcn2 deficiency, however, did not affect acute nociceptive pain. Lcn2-deficient mice showed significantly less microglial activation and proalgesic chemokine (CCL2 and CXCL1) production in the spinal cord after SNI than wild-type mice, and recombinant LCN2 protein induced the expression of these chemokines in cultured neurons. Furthermore, the expression of LCN2 and its receptor was detected in neutrophils and macrophages in the sciatic nerve following SNI, suggesting the potential role of peripheral LCN2 in neuropathic pain. Taken together, our results indicate that LCN2 plays a critical role in the development of pain hypersensitivity following peripheral nerve injury and suggest that LCN2 mediates neuropathic pain by inducing chemokine expression and subsequent microglial activation.

Keywords: Chemokines; Glia; Nerve; Neuroinflammation; Neuropathic Pain; Pain; Spinal Cord.

Figures

FIGURE 1.
FIGURE 1.
Expression of Lcn2 mRNA in the spinal cord after SNI. A–C, the expression of Lcn2 mRNA in the spinal cord or DRG after SNI was assessed using traditional RT-PCR (A, left, and B and C) or real-time PCR (A, right). Lcn2 mRNA levels in ipsilateral region of the dorsal horn of the spinal cord were significantly increased at 1–3 days after SNI and then decreased to basal levels (A). The mRNA levels of Lcn2 in ipsilateral dorsal horn were significantly higher than those in contralateral sides in three animals (contra- or ipsi-1, -2, and -3) at 3 days after SNI (B). The mRNA levels of Lcn2 in the DRG were not significantly changed by SNI at any time point (C). Results are representative of either three independent experiments or means ± S.E. *, p < 0.05 versus the control group or contralateral versus ipsilateral side, n = 3. Results of densitometric analyses normalized to GAPDH are shown either below or to the right of the gels.
FIGURE 2.
FIGURE 2.
Immunolocalization of LCN2 in the spinal cord after SNI. A, a low level of LCN2 immunoreactivity was detected in the spinal cord of naive control mice (panels a and g). At 3 days after SNI, LCN2 immunoreactivity was detected in the contralateral (Contra) dorsal and ventral horn (panels b and h) and in the ipsilateral (Ipsi) dorsal and ventral horn (panels c and i). Scale bars, 100 μm. Double immunostaining showed that LCN2 (green) in the dorsal (panels d–f) and ventral horn (panels j–l) of the spinal cord was co-localized with NeuN (red), a neuronal marker. Arrows indicate examples of doubly labeled cells. Inset in panel f, high magnification image showing doubly labeled cells in the ipsilateral dorsal horn. Scale bars, 100 μm. B, double immunostaining showed that LCN2 (green) in the ipsilateral dorsal horn of the spinal cord at 3 days after SNI was not co-localized with Iba-1 (red) (panel a) or GFAP (red) (panel b). Scale bars, 100 μm. Results are a representative of more than three independent experiments.
FIGURE 3.
FIGURE 3.
Effect of LCN2 on mechanical allodynia. A, SNI induced a significant decrease in paw withdrawal threshold in wild-type (WT) mice, whereas SNI-induced mechanical allodynia was significantly inhibited at 1–3 days in Lcn2-deficient (KO) mice. Data are means ± S.E. *, p < 0.05 as compared with the ipsilateral hind paw of wild-type mice, n = 5–6. Intrathecal injection of LCN2 protein (0.1 and 1 μg) into naive mice induced mechanical allodynia. Contra, contralateral side; Ipsi, ipsilateral side. B, mechanical allodynia increased rapidly 3 h after LCN2 (1 μg) administration and was maintained for 3 days. The results shown are means ± S.E. *, p < 0.05 versus the vehicle (PBS) group, n = 4–5.
FIGURE 4.
FIGURE 4.
Effect of LCN2 on the activations of microglia and p38 MAPK after SNI. A, Iba-1 and phospho-p38 MAPK immunoreactivities were detected in the spinal dorsal (panels a–d) and ventral horn (panels i–l) of wild-type (WT) mice and in the spinal dorsal (panels e–h) and ventral horn (panels m–p) of Lcn2-deficient (KO) mice at 3 days after SNI. Scale bar, 100 μm. Results are representative of more than three independent experiments. B and C, quantified immunohistochemical data for Iba-1 and phospho-p38 MAPK IR are shown in the bar graphs, which display the number of Iba-1-positive cells (B) or phospho-p38 MAPK-positive cells (C) in the dorsal and ventral horn of the spinal cord at 3 days after SNI. Results are presented as means ± S.E. *, p < 0.05 versus the contralateral side; #, p < 0.05, the ipsilateral side of WT versus KO mice, n = 3. Contra, contralateral side; Ipsi, ipsilateral side.
FIGURE 5.
FIGURE 5.
Effect of LCN2 on chemokine expression after SNI. A and B, relative mRNA expression of Ccl2 (A) and Cxcl1 (B) in the contralateral or ipsilateral side of the spinal cord at 2 days after SNI was evaluated by real-time RT-PCR. The expression of both chemokines was significantly attenuated in the spinal cord of Lcn2-deficient (KO) mice after SNI. Results are presented as means ± S.E. *, p < 0.05 versus the contralateral side (Contra); #, p < 0.05, the ipsilateral side (Ipsi) of WT versus KO mice, n = 3.
FIGURE 6.
FIGURE 6.
Immunolocalization of 24p3R (the LCN2 receptor) in spinal cord after SNI. A–C and G–I, 24p3R immunoreactivity in naive control animals (A and G), in the contralateral (Contra) dorsal and ventral horn of the spinal cord (B and H) and in the ipsilateral (Ipsi) dorsal and ventral horn (C and I) of the spinal cord at 3 days after SNI. Scale bars, 100 μm. D–F and J–L, double immunostaining showed that 24p3R (green) in the dorsal (D--F) and ventral horn (J–L) of the spinal cord was co-localized with NeuN (red) or Iba-1 (red), but not with GFAP (red). High magnification images (insets in D, E, and K) indicate doubly labeled cells in the ipsilateral dorsal (D and E) or ventral horn (K), respectively. Arrows indicate examples of doubly labeled cells. Scale bars, 100 μm. Results are representative of more than three independent experiments.
FIGURE 7.
FIGURE 7.
LCN2 induction of chemokine gene expression in primary cortical neurons. Primary cortical neurons were incubated with recombinant LCN2 protein (1 or 10 μg/ml) for 3, 6, or 12 h, and total RNA was isolated for real-time RT-PCR. A–E, the mRNA levels of chemokines, such as Ccl2 (A), Ccl3 (B), Ccl5 (C), Cxcl1 (D), and Cxcl10 (E) were determined. Results are means ± S.E. *, p < 0.05 versus the denatured LCN2 protein-treated group (Control) at each time point (n = 3).
FIGURE 8.
FIGURE 8.
Immunolocalization of LCN2 in the sciatic nerve after SNI. A–L, LCN2 immunoreactivity was examined at the site of ligature (A–D) or proximal (E–H) and distal (I–L) sites of the sciatic nerve injury at 3 days after SNI. High magnification image (inset in E) indicates LCN2-positive cells in the sciatic nerve. Double immunostaining showed that LCN2 (green or red) in the sciatic nerve was co-localized with Ly6G (green; C, G, and K), but not with Iba-1 (red; B, F, and J) or 24p3R (red; D, H, and L). High magnification image (inset in G) indicates doubly labeled cells in the sciatic nerve. S.N, sciatic nerve. Scale bars, 100 μm. Results are representative of more than three independent experiments.
FIGURE 9.
FIGURE 9.
The involvement of LCN2 in the development of pain hypersensitivity following peripheral nerve injury. LCN2 synthesized in spinal neurons after peripheral nerve injury may be released into the extracellular space and bind to 24p3R, which is expressed on the surfaces of neurons and microglia (and astrocytes) in the spinal cord. We surmise that after binding to its receptor, LCN2 could induce the expression and release of chemokines from spinal neurons or other cell types. These chemokines may in turn activate spinal microglia, and thus, facilitate neuroinflammation and the trafficking of other glial and inflammatory cells, ultimately leading to pain sensitization in the spinal cord. Taken together, our findings suggest that the LCN2-chemokine axis plays a central role in the development of pain hypersensitivity under conditions such as neuropathic pain.

Similar articles

See all similar articles

Cited by 8 articles

See all "Cited by" articles

Publication types

MeSH terms

Feedback