Tissue plasminogen activator contributes to morphine tolerance and induces mechanical allodynia via astrocytic IL-1β and ERK signaling in the spinal cord of mice

Abstract

Accumulating evidence indicates that activation of spinal cord astrocytes contributes importantly to nerve injury and inflammation-induced persistent pain and chronic opioid-induced antinociceptive tolerance. Phosphorylation of extracellular signal-regulated kinase (pERK) and induction of interleukin-1 beta (IL-1β) in spinal astrocytes have been implicated in astrocytes-mediated pain. Tissue plasminogen activator (tPA) is a serine protease that has been extensively used to treat stroke. We examined the potential involvement of tPA in chronic opioid-induced antinociceptive tolerance and activation of spinal astrocytes using tPA knockout (tPA(-/-)) mice and astrocyte cultures. tPA(-/-) mice exhibited unaltered nociceptive pain and morphine-induced acute analgesia. However, the antinociceptive tolerance, induced by chronic morphine (10mg/kg/day, s.c.), is abrogated in tPA(-/-) mice. Chronic morphine induces tPA expression in glial fibrillary acidic protein (GFAP)-expressing spinal cord astrocytes. Chronic morphine also increases IL-1β expression in GFAP-expressing astrocytes, which is abolished in tPA-deficient mice. In cultured astrocytes, morphine treatment increases tPA, IL-1β, and pERK expression, and the increased IL-1β and pERK expression is abolished in tPA-deficient astrocytes. tPA is also sufficient to induce IL-1β and pERK expression in astrocyte cultures. Intrathecal injection of tPA results in up-regulation of GFAP and pERK in spinal astrocytes but not up-regulation of ionized calcium binding adapter molecule 1 in spinal microglia. Finally, intrathecal tPA elicits persistent mechanical allodynia, which is inhibited by the astroglial toxin alpha-amino adipate and the MEK (ERK kinase) inhibitor U0126. Collectively, these data suggest an important role of tPA in regulating astrocytic signaling, pain hypersensitivity, and morphine tolerance.

Keywords: DRGs; ERK; ERK kinase; FBS; GAPDH; GFAP; IBA-1; IL-1β; L-2-AA; LTP; MEK; MMP-2; PWL; WT; acute opioid analgesia; chronic morphine exposure; dorsal root ganglia; extracellular signal-regulated kinase; extracellular signal-regulated kinase (ERK); fetal bovine serum; glial fibrillary acidic protein; glyceraldehyde3-phosphate dehydrogenase; interleukin-1 beta; interleukin-1 beta (IL-1β); ionized calcium binding adapter molecule 1; l-2-Aminoadipic acid; long-term potentiation; matrix metalloprotease-2; pERK; paw withdrawal latency; phosphoERK; protease; tPA; tPA knockout mice; tissue plasminogen activator; wild-type.

Figure 1. tPA^−/− mice display normal nociceptive pain, unaltered acute morphine analgesia, but diminished morphine tolerance

(A) Heat and mechanical sensitivity in WT and tPA^−/− mice. n=5 mice. Heat and mechanical pain sensitivity was measured by radiant heat (Hargreaves) and von Frey hairs, respectively. (B) Acute morphine (10 mg/kg, s.c.)-induced analgesia in WT and tPA^−/− mice. n=5 mice. Morphine analgesia was determined by tail flick latency in the hot water immersion test. (C) Chronic morphine (10 mg/kg, s.c., daily for 10 days)-induced antinociceptive tolerance in WT and tPA^−/− mice. *P<0.05, compared with WT mice at the same time point, n=6 mice, t-test. Morphine analgesia was determined by tail flick latency at 30 min after the daily morphine injection.

Figure 2. Chronic morphine treatment induces tPA expression in spinal cord astrocytes

(A, B) Western blotting showing tPA expression in the DRG and spinal cord dorsal horn following chronic morphine exposure (5 d). Low panels, intensity of tPA bands. *P<0.05, compared to control (Saline), t-test, n=5 mice. (C) Immunohistochemistry showing chronic morphine-induced tPA increase in the superficial dorsal horn. Low panel, intensity of tPA staining in the superficial dorsal horn. *P<0.05, t-test, compared to control (Saline), n=5 mice. Scale bar, 100 μm. (D) Confocal images showing colocalization of tPA and GFAP after chronic morphine. Scale bars, 100 μm (low magnification image) and 25 μm (high magnification images). (E) Real-time qRT-PCR analysis showing time courses of tPA, GFAP and IL-1β mRNA expression in the dorsal horn, before and 3, 5 and 7 days after chronic morphine exposure. *P<0.05, compared to control (Ctrl), t-test, n=4 mice. Spinal cord (dorsal part) and DRG tissues were collected 2 h after the 3^th, 5^th, or 7^th daily morphine injection (10 mg/kg, s.c.).

Figure 3. Chronic morphine induces IL-1β expression in spinal cord astrocytes via tPA

(A) Western blot showing GFAP expression in the spinal cord dorsal horn of WT and tPA^−/− mice following chronic morphine exposure (5 d). (B) Quantification of the GFAP bands. *P<0.05, compared to control (Saline), t-test, n=5 mice. (C) GFAP immunostaining in the dorsal horn of WT and tPA^−/− mice after chronic morphine treatment. The boxes are enlarged in D. Scale, 100 μM. (D) GFAP and IL-1β double staining in the dorsal horn of WT and tPA^−/− mice after chronic morphine treatment. Arrows indicate the double-labeled cells. The cell indicated by blue arrow is enlarged in the small insert. Note that chronic morphine induces IL-1β expression in astrocytes. Scale, 25 μM. (E) Intensity of GFAP staining in the superficial dorsal horn (laminae I–III). *P<0.05, compared to WT control, ANOVA, n=5 mice. (F) Number of IL-1β/GFAP double-labeled cells in the superficial dorsal horn (laminae I–III). *P<0.05, compared to WT control, ^#P<0.05, compared to morphine WT group, ANOVA, n=5 mice.

Figure 4. Chronic morphine induces IL-1β and pERK expression in astrocyte cultures via tPA

(A) Western blot showing tPA expression following chronic morphine treatment (100 μM, 5 days) in astrocyte cultures from WT mice. The line under the gel indicates the two parts are from the same gel but not adjacent. Right panel, intensity of tPA bands. *P<0.05, compared to Control (saline), t-test, n=4 cultures. (B) Western blots showing pERK and IL-1β expression in astrocytes from WT (left blot) and tPA^−/− (right blot) mice before and after morphine treatment. (C) Intensity of pERK (p42/44) and IL-1β bands in astrocytes from WT (left graph) and tPA^−/− (right graph) mice before and after morphine treatment. Morphine increases pERK and IL-1β expression in wild-type but not tPA-deficient astrocytes. *P<0.05, compared to control; ^#P<0.05; n=4 cultures. (D) tPA (50 ng/ml, 1 day) induces pERK and IL-1β expression in WT astrocytes. *P<0.05, compared with control, n=4 cultures.

Figure 5. Intrathecal injection of tPA elicits persistent mechanical allodynia

(A) A single intrathecal injection of tPA (10 μg) induces rapid and persistent mechanical allodynia, as assessed by von Frey hairs. *P<0.05, vs. PBS control, t-test. n=4–7 mice. (B) Intrathecal injection of tPA (10 μg) does not produce heat hyperalgesia, as measured by radiant heat test (Hargreaves). P>0.05, vs. vehicle control, t-test, n=4–7 mice.

Figure 6. Intrathecal injection of tPA induces pERK in astrocytes of the spinal cord dorsal horn

(A) Immunofluorescence of IBA-1, GFAP, and pERK in the dorsal horn 2 days after intrathecal injection of tPA (10 μg) and saline. The white boxes are enlarged in the panels below. Scale bars, 100 μm. (B–D) Intensity of immunofluorescence of IBA-1 (B), GFAP (C) and pERK (D) in the superficial dorsal horn (laminae I-III) 2 days after intrathecal tPA (10 μg) and vehicle. *P<0.05, vs. vehicle control, t-test. n=5 mice. (E) Confocal images showing the double staining of pERK and GFAP in the superficial dorsal horn 2 days after intrathecal tPA (10 μg). Arrows indicate the double-labeled cells. Scale, 25 μm.

Figure 7. tPA-induced mechanical allodynia is attenuated by intrathecal inhibition of astrocytes and ERK

(A, B) Partial reversal of intrathecal tPA (10 μg)-induced mechanical allodynia by intrathecal injection of the astrocyte toxin L-2-AA (A) and the MEK (ERK kinase) inhibitor U0126 (B). *P<0.05, vs. saline control, t-test. n=5 mice.