Distinct roles of matrix metalloproteases in the early- and late-phase development of neuropathic pain

Abstract

Treatment of neuropathic pain, triggered by multiple insults to the nervous system, is a clinical challenge because the underlying mechanisms of neuropathic pain development remain poorly understood. Most treatments do not differentiate between different phases of neuropathic pain pathophysiology and simply focus on blocking neurotransmission, producing transient pain relief. Here, we report that early- and late-phase neuropathic pain development in rats and mice after nerve injury require different matrix metalloproteinases (MMPs). After spinal nerve ligation, MMP-9 shows a rapid and transient upregulation in injured dorsal root ganglion (DRG) primary sensory neurons consistent with an early phase of neuropathic pain, whereas MMP-2 shows a delayed response in DRG satellite cells and spinal astrocytes consistent with a late phase of neuropathic pain. Local inhibition of MMP-9 by an intrathecal route inhibits the early phase of neuropathic pain, whereas inhibition of MMP-2 suppresses the late phase of neuropathic pain. Further, intrathecal administration of MMP-9 or MMP-2 is sufficient to produce neuropathic pain symptoms. After nerve injury, MMP-9 induces neuropathic pain through interleukin-1beta cleavage and microglial activation at early times, whereas MMP-2 maintains neuropathic pain through interleukin-1beta cleavage and astrocyte activation at later times. Inhibition of MMP may provide a novel therapeutic approach for the treatment of neuropathic pain at different phases.

Figure 1. Upregulation of MMP-9 in the DRG after spinal nerve ligation (SNL)

(a) Gelatin zymography shows a time course of MMP-9 and MMP-2 activity in the injured L5 DRG of SNL rats. Right panel shows intensity of specific MMP bands, expressed as fold change over non-injured control. * p<0.05, compared to control (n=5). (b) MMP-9 Western blotting in the L5 DRG of sham and SNL rats. Right panel, quantification of MMP-9 band intensity (* p<0.05, n=3). (c) MMP-9 immunohistochemistry in the L5 DRG of control and SNL rats. Note that MMP-9 is expressed in DRG neurons. Scale, 50 μm.

Figure 2. MMP-9 is both required and sufficient for producing neuropathic pain symptom

(a) Persistent infusion of MMP-9 inhibitor delays the development of mechanical allodynia in SNL rats. The inhibitor or vehicle (20% DMSO) was intrathecally infused via an osmotic pump (0.5 μl/h, 7 days) starting 2 days before SNL (* p<0.05, compared to corresponding vehicle control, n=6). BL indicates baseline. (b) Reversal of mechanical allodynia by endogenous MMP-9 inhibitor TIMP-1 in SNL rats (* p<0.05, compared to saline, n=6). (c) Pretreatment of MMP-9 siRNA (2 × 5 μg, i.t.) delays the development of mechanical allodynia in SNL rats (* p<0.05, compared to respective mismatch control RNA, n=9). Insert, gelatin zymography showing knockdown of MMP-9 but not MMP-2 by MMP-9 siRNA in the DRG on SNL day 1. (d) Intrathecal administration of MMP-9 induces rapid but reversible mechanical allodynia in rats (* p<0.05, compared to saline control, n=12). (e) MMP-9 null mice show a reduction in SNL-induced spontaneous pain at early times (* p<0.05, compared to wild type mice, n=6). Spontaneous pain was evaluated by counting the number of flinches of affected hindpaws over a 2-minute period. (f) MMP-9 null mice show a reduction of mechanical allodynia at early times after SNL (* p<0.05, compared to wild type mice, n=6). Insert, gelatin zymography showing absence of MMP-9 in MMP-9 null mice.

Figure 3. MMP-9 produces neuropathic pain via microglia activation and IL-1β signaling

(a) Immunostaining of microglia surface marker OX-42 (CD11b) in rat dorsal horn after intrathecal saline or MMP-9 (1 pmol). Lower panel, high magnification of microglia. Scales, 50 μm. Right panel, number of OX-42+ microglia following treatment of MMP-9 and MMP-9 siRNA (2 × 5 μg) in rats (* p<0.05, n=4). (b) Immunostaining of phospho-p38 (P-p38) in the dorsal horn after intrathecal MMP-9 (1 pmol). Low panel, colocalization of P-p38 and OX-42 in spinal microglia after MMP-9 treatment. Scales, 50 (upper) and 10 μm (lower). Right panel, number of P-p38+ cells in the dorsal horn following treatment of MMP-9 and MMP-9 siRNA (* p<0.01, n=4). (c) p38 inhibitor FR167653 (100 nmol, i.t.) blocks mechanical allodynia induced either by MMP-9 (1 pmol) or SNL (* p<0.05, n=6). (d) Left panel: intrathecal MMP-9 (1 pmol) induces IL-1β cleavage in the DRG. Right panel, IL-1β cleavage after SNL is reduced in MMP-9 null mice. (e) ELISA shows DRG IL-1β levels after treatment of MMP-9, SNL, and MMP-9 siRNA (*, p<0.05, n=4-6). (f) Mechanical allodynia induced by MMP-9 and SNL is reversed by IL-1β neutralizing antibody (5 μg, i.t., * p<0.05, n=6). (g) Schematic representation of MMP-9-triggered sequential events for the genesis of neuropathic pain. MMP-9 upregulation after nerve injury increases IL-1β cleavage in the DRG and spinal cord. MMP-9 and IL-1β are transported to spinal central terminals. Dashed arrows indicate previously known events. A positive feedback loop between IL-1β and p38 can enhance the production of IL-1β, leading to increased pain sensitivity.

Figure 4. MMP-2 upregulation after SNL maintains neuropathic pain via IL-1β signaling and ERK activation in spinal astrocytes

(a) Gelatin zymography reveals MMP-2 upregulation in rat DRGs 10 days after SNL. Low panel, intensity of MMP bands (* p<0.05, n=3). Right panel, MMP-2 expression in DRG satellite cells. Scale, 50 μm. (b) Gelatin zymography shows MMP-2 upregulation in rat dorsal horn after SNL. Lower panel, intensity of MMP-2 bands (* p<0.05, compared to naive control, n=5). Upper right panel reveals MMP-2 expression in the ipsilateral superficial dorsal horn. Scale, 50 μm. Lower right panel shows double staining of MMP-2 with GFAP and OX-42. Scale, 10 μm. (c) Reversal of SNL-induced mechanical allodynia by TIMP-2 (4.5 pmol, i.t.) in SNL rats (* p<0.05, n=6). (d) Repeated daily injections of a synthetic MMP-2 inhibitor (10 nmol, i.t.) persistently attenuates SNL-induced allodynia in mice (* p<0.05, compared to corresponding vehicle, n=5). (e) Reversal of SNL-induced allodynia by the MMP-2 inhibitor (10 nmol, i.t.) in both MMP-9 knockout and wild type mice. Note that MMP-2 inhibitor is more effective in knockout mice (* p<0.05, compared to respective pre-injection baseline, n=5). (f) Reversal of SNL-induced mechanical allodynia by MMP-2 siRNA2 (2×2 μg, i.t.) in mice (* P<0.05, n=6). (g) MMP-2 siRNA2 treatment (2 × 2 μg, i.t.) inhibits IL-1β cleavage in the DRG and dorsal horn (78% and 29% reduction of 17 kD band, respectively, p<0.05) and ERK activation (39% reduction of pERK1/2 bands, p<0.05) in the dorsal horn of SNL mice (day 10).