Hevin/Sparcl1 drives pathological pain through spinal cord astrocyte and NMDA receptor signaling

Abstract

High endothelial venule protein/SPARC-like 1 (hevin/Sparcl1) is an astrocyte-secreted protein that regulates synapse formation in the brain. Here we show that astrocytic hevin signaling plays a critical role in maintaining chronic pain. Compared with WT mice, hevin-null mice exhibited normal mechanical and heat sensitivity but reduced inflammatory pain. Interestingly, hevin-null mice have faster recovery than WT mice from neuropathic pain after nerve injury. Intrathecal injection of WT hevin was sufficient to induce persistent mechanical allodynia in naive mice. In hevin-null mice with nerve injury, adeno-associated-virus-mediated (AAV-mediated) re-expression of hevin in glial fibrillary acidic protein-expressing (GFAP-expressing) spinal cord astrocytes could reinstate neuropathic pain. Mechanistically, hevin is crucial for spinal cord NMDA receptor (NMDAR) signaling. Hevin-potentiated N-Methyl-D-aspartic acid (NMDA) currents are mediated by GluN2B-containing NMDARs. Furthermore, intrathecal injection of a neutralizing Ab against hevin alleviated acute and persistent inflammatory pain, postoperative pain, and neuropathic pain. Secreted hevin that was detected in mouse cerebrospinal fluid (CSF) and nerve injury significantly increased CSF hevin abundance. Finally, neurosurgery caused rapid and substantial increases in SPARCL1/HEVIN levels in human CSF. Collectively, our findings support a critical role of hevin and astrocytes in the maintenance of chronic pain. Neutralizing of secreted hevin with monoclonal Ab may provide a new therapeutic strategy for treating acute and chronic pain and NMDAR-medicated neurodegeneration.

Keywords: Neurological disorders; Neuroscience.

Figure 1. Baseline pain, inflammatory pain, and neuropathic pain in WT and hevin-KO mice.

There are no significant differences in mechanical and thermal pain threshold between WT and hevin-KO male mice, as shown in von Frey test (A), radiant heat test (B), hot plate test (C) and tail immersion test (D). P > 0.05, unpaired Student’s t test, n = 11 mice/group. (E) Formalin-induced acute inflammatory pain was significantly reduced in hevin-KO male mice. Left, time-course of licking and flinching behavior following intraplantar injection of 5% formalin. Right, formalin-induced Phase I (1–10 min) and Phase II (10–45 min) responses. *P < 0.05, 2-way ANOVA followed by Bonferroni’s post hoc test. n = 6 mice/group. (F) Mechanical allodynia, induced by intraplantar injection of carrageenan, recovered faster in hevin-KO male mice than in WT male mice. Arrow indicates the time of carrageenan injection. *P < 0.05, **P < 0.01, ***P < 0.001, compared with baseline (BL) group; ^#P < 0.05, 2-way ANOVA followed by Bonferroni’s post hoc test. n = 5 mice/group. Data shown as mean ± SEM. (G) Hevin-KO male mice recovered faster from CCI-induced persistent mechanical allodynia than WT male mice. ***P < 0.001 compared with BL group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, 2-way ANOVA followed by Bonferroni’s post hoc test. n = 11 mice/group. (H) Ongoing pain 3 weeks after CCI in WT and hevin-KO male mice were tested using a 2-chamber CPP. Ongoing pain was present in WT mice but absent in hevin-KO mice following clonidine treatment (10 μg, i.t.). *P < 0.05, unpaired Student’s t test, n = 6 mice/group. All data are shown as mean ± SEM.

Figure 2. Intrathecal administration of hevin but not hevin-ΔDE (10 μg, i.t.) decreases PWT in naive mice and mice with nerve injury and enhances spontaneous pain by formalin.

(A) Intrathecal injection of WT hevin but not mutant hevin (hevin-ΔDE) induced mechanical allodynia in naive mice lasting more than 3 days. n = 5 mice/group. **P < 0.01, ***P < 0.001 compared with BL group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, 2-way ANOVA followed by Bonferroni’s post hoc test. Arrow indicates hevin injection on day 0. (B) Repeated intrathecal injections of WT hevin but not mutant hevin (hevin-ΔDE), given at 12 and 13 days after CCI induced further exacerbated mechanical allodynia. n = 5 mice/group. *P < 0.05, **P < 0.01, compared with CCI-12d baseline group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, 2-way ANOVA followed by Bonferroni’s post hoc test. Arrows indicate the time of hevin injections. (C–E) Intrathecal injection of WT hevin but not mutant hevin (hevin-ΔDE) enhances formalin-induced licking and flinching on Phase 2 (10–45 min) and Phase 2⁺ (45–60 min) but not Phase 1 (0–10 min) in both male C and female D mice. *P < 0.05, ***P < 0.001, compared with hevin-ΔDE group, 2-way ANOVA followed by Bonferroni’s post hoc test. All data are shown as mean ± SEM.

Figure 3. RNAScope images showing Sparcl1 expression in excitatory and inhibitory neurons in the SDH of VGLUT2:Ai9 mice.

(A) RNAScope images show colocalization of Sparcl1 (red) with VGLUT2⁺ excitatory neurons (green) and Slc32a1⁺ inhibitory neurons (blue). Top, merged low-magnification image. Scale bar: 50 μm. Bottom, merged and single-channel images enlarged from the box in the top panel. Filled arrows show Sparcl1⁺/VGLUT2⁺ excitatory neurons, open arrows show Sparcl1⁺/Slc32a1⁺ inhibitory neurons, and arrows with cross show only Sparcl1⁺ cells. Scale bar: 20 μm. (B) Quantification of the percentage of Sparcl1⁺ cells expressing VGLUT2 or Slc32a1 and the percentage of VGLUT2⁺ or Slc32a1⁺ cells expressing Sparcl1 in the SDH. n = 6 spinal cord sections from 3 mice. Data are shown as mean ± SEM.

Figure 4. Expression of hevin in spinal astrocytes by intraspinal hevin-AAV reinstates neuropathic pain in hevin-KO mice.

(A) Double immunostaining of hevin (green) and GFAP (red) in SDH. Note hevin is primarily colocalized with GFAP. Scale bars: 100 μm (left); 20 μm (right). The box is enlarged in the right panels. (B) Absence of hevin immunostaining in SDH in hevin-KO mice. Scale bar: 100 μm. (C) Paradigm for measuring mechanical allodynia in hevin-KO mice with intraspinal microinjection of hevin-AAV and hevinΔDE-AAV, given 6 days before CCI. (D) SDH microinjection of AAV-induced reduction in PWT in naive hevin-KO mice. After CCI, mechanical allodynia was significantly more prolonged in hevin-AAV–treated mice than hevinΔDE-AAV–treated mice. n = 6 mice/group. *P < 0.05, 2-way ANOVA followed by Bonferroni’s post hoc test. Green and red arrows indicate the time of virus injection and nerve injury, respectively. (E) Paradigm for measuring mechanical allodynia in hevin-KO mice with intraspinal microinjection of hevin-AAV and hevinΔDE-AAV, given 2 days after CCI. (F) SDH microinjection of hevin-AAV, given after nerve injury, significantly enhanced and prolonged mechanical allodynia in hevin-KO mice vs. hevinΔDE-AAV–treated mice. n = 6 mice/group. *P < 0.05, **P < 0.01, ***P < 0.001, 2-way ANOVA followed by Bonferroni’s post hoc test. Arrows indicate the time of virus injection and nerve injury. (G) Triple immunostaining of Myc (red), hevin (green), and GFAP (blue) in SDH in hevin-KO mice, 24 days after the ipsilateral SDH hevin-AAV injection. Note hevin expression is absent in the contralateral SDH of hevin-KO mice. Scale bar: 100 μm. (H) Enlarged images in the box of F panel G, with additional merged images for Myc/hevin, Myc/GFAP, and hevin/GFAP. Note hevin⁺ astrocytes also Myc⁺/GFAP⁺ in superficial SDH. Scale bar: 20 μm. All data are shown as mean ± SEM.

Figure 5. Hevin regulates NMDA-evoked pain and enhances NMDA currents in SDH neurons.

(A) The duration of mechanical allodynia, induced by intrathecal injection of NMDA (3 nmol), is significantly shorter in hevin-KO mice than in WT mice. n = 6 mice/group. *P < 0.05, **P < 0.01, ***P < 0.001, compared with BL group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, 2-way ANOVA followed by Bonferroni’s post hoc test. Arrow indicates the time of NMDA injection. (B and C) Left: representative traces of inward currents in WT and hevin-KO mice, induced by NMDA (100 μM, 3 sec) via bath application. Note smaller NMDA currents in hevin-KO mice and different effects of RO25-6091 (GluN2B antagonist) and TCN-201 (GluN2A antagonist). Right: amplitude of NMDA-induced currents. n = 9, 10 neurons per group (shown in each column). *P < 0.05, 1-way ANOVA followed by Bonferroni’s post hoc test. (D and E) Left: representative traces of inward currents in ACSF- and hevin-treated (100 ng/mL, 4 min) spinal cord slices, induced by NMDA (100 μM, 3 sec) via bath application. Note different effects of Ro25-6091 and TCN-201. Right: amplitude of NMDA-induced currents. n = 7 neurons per group. *P < 0.05, **P < 0.01, ***P < 0.001, 1-way ANOVA followed by Bonferroni’s post hoc test. Data are shown as mean ± SEM.

Figure 6. Anti-hevin monoclonal Ab 12:54 reduces inflammatory, postoperative, and neuropathic pain in WT mice.

(A) ELISA analysis showing increased hevin level in the CSF 14 days after CCI. n = 5 mice/group. *P < 0.05, unpaired Student’s t test. (B) Left, time course of formalin-induced pain in WT male mice treated with intrathecal anti-hevin 12:155 monoclonal Ab (control Ab, 10 μg) or anti-hevin 12:54 monoclonal Ab (function blocking Ab, 10 μg). n = 5 mice per group. Right, formalin-induced Phase 1 and Phase 2 responses. *P < 0.05, unpaired Student’s t test. (C) Intrathecal injection of anti-hevin 12:54 Ab (10 μg), given 3 days after CFA injection, reduced CFA-induced mechanical allodynia for 5 hours. Arrows indicate the time of Ab injection. n = 5 mice/group. ^#P < 0.05, ^##P < 0.01 versus corresponding BL group; *P < 0.05 versus anti-hevin 12:155 group, 2-way ANOVA followed by Bonferroni’s post hoc test. (D) Intrathecal injection of anti-hevin 12:54 Ab (10 μg) given 3 hours after plantar incision, reduced incision-induced mechanical allodynia for 5 hours in male and female mice. Arrows indicate the time of Ab injection. n = 10 mice/group. ^###P < 0.001 versus corresponding BL group; ***P < 0.001 versus anti-hevin 12:155 group, 2-way ANOVA followed by Bonferroni’s post hoc test. (E and F) Intrathecal injection of anti-hevin 12:54 Ab (10 μg), given 7 days E and 21 days F after nerve injury, reduced CCI-induced mechanical allodynia for 5 hours. Arrows indicate the time of Ab injection. n = 5–6 mice/group. ^#P < 0.05 versus corresponding BL group; ^§P < 0.05, ^§§P < 0.01 versus corresponding baseline at CCI 7 days or CCI 21 days; *P < 0.05, **P < 0.01, 2-way ANOVA followed by Bonferroni’s post hoc test. Data are shown as mean ± SEM.

Figure 7. Intracranial surgery increases hevin levels in human CSF samples.

(A and B) ELISA analysis showing increased hevin levels in human CSF 12 hours after intracranial surgery. The same data were presented unpaired A and paired B. (C) Bicinchoninic acid (BCA) protein assay showing total protein level increased in human CSF 12 hours after surgery. (D) Fold changes of normalized hevin level in human CSF 12 hours after surgery, normalized to total protein changes. n = 10 patients. **P < 0.01, ***P < 0.001, paired Student’s t test. Data shown as mean ± SEM.