Fibronectin inhibits chronic pain development after spinal cord injury

Abstract

Chronic pain following spinal cord injury (SCI) is a highly prevalent clinical condition that is difficult to treat. Using both von Frey filaments and radiant infrared heat to assess mechanical allodynia and thermal hyperalgesia, respectively, we have demonstrated that a one-time injection of fibronectin (50 μg/mL) into the spinal dorsal column (1 μL/min each injection for a total of 5 μL) immediately after SCI inhibits the development of mechanical allodynia (but not thermal hyperalgesia) over an 8-month observation period following spinal cord dorsal column crush (DCC). DCC will only induce mechanical Allodynia, but not thermal hyperalgesia or overt motor deficits. By applying various fibronectin fragments as well as competitive inhibitors, these effects were shown to be dependent on the connecting segment-1 (CS-1) motif of fibronectin. Furthermore, we found that acute fibronectin treatment diminished inflammation and blood-spinal cord barrier permeability, which in turn leads to enhanced fiber sparing and sprouting. In particular, the reduction of serotonin (5-HT) in the superficial dorsal horn, an important descending brainstem system in the modulation of pain, was blocked with fibronectin treatment. We conclude that treatment of SCI with fibronectin preserves sensory regulation and prevents the development of chronic allodynia, providing a potential therapeutic intervention to treat chronic pain following SCI.

FIG. 1.

Fibronectin treatment blocks mechanical allodynia, but not thermal hyperalgesia in dorsal column crush (DCC) rats. (A) Both CS-1 (DCC+CS-1, n=8) and fibronectin (DCC+FN, n=8) treatment resulted in a complete block of mechanical allodynia compared to rats treated with vehicle only (DCC+vehicle, n=10). (B) The hindpaw withdrawal latencies were not significantly influenced by DCC or fibronectin treatment. A thin line was added to indicate the pre-DCC baseline values as compared with later time points to showcase the development of allodynia.

FIG. 2.

Different fibronectin fragments exerted different effects on allodynia following dorsal column crush (DCC), and the competitive inhibitor N-1765 blocked fibronectin's effects. (A) Treatment with additional two fibronectin fragments including a 50-kDa (n=3) and a 120-kDa fragment (n=3) also inhibited DCC-induced mechanical allodynia development, but to a lesser extent compared to the groups treated with connecting segment-1 (CS-1, n=5) or fibronectin (FN, n=4). (B) N-1765 treatment caused significant decreases in hindpaw withdrawal thresholds from 1 week after DCC (*versus vehicle-treated rats; ^xversus N-1765-treated rats).

FIG. 3.

Fibronectin treatment decreased dorsal column crush (DCC)-induced increases in blood–spinal cord barrier (BSCB) permeability, and the immunoreactivity of glial fibrillary acidic protein (GFAP) and ED1. (A) Rats with DCC were injected with fibronectin (DCC+FN, n=4). BSCB permeability to sodium fluorescein (NaF) was assessed in the tissues including at the epicenter (1-cm length of spinal cord surrounding the epicenter of lesion), and at the areas (1-cm-long) both rostral and caudal to the lesion of the spinal cord at 2 and 9 days after DCC. BSCB permeability changes are presented as the mean±standard deviation of NaF uptake in the tissues (μg NaF/μg tissue). Representative immunolabeling of GFAP (A–C, Normal, DCC, and DCC+FN), and ED1 (E–G) in the area caudal to the lesion demonstrated upregulation of GFAP (C) and ED1 (F), compared to normal rats (A and E) at 3 days after DCC. The graphs illustrate a significant increase after DCC (*versus normal rats), but a decrease with fibronectin treatment (^xversus vehicle-treated DCC rats) in GFAP (D) and ED1 (H) immunoreactivity both rostral and caudal to the lesion (n=3 each group; N, normal rats; DC, vehicle-treated DCC rats; DC+FN, fibronectin-treated DCC rats; scale bar=100 mm).

FIG. 4.

Serotonin (5-HT) immunoreactivity in the superficial dorsal horn was changed by dorsal column crush (DCC) and fibronectin treatment. Photomicrographs of 5-HT immunofluorescence from representative sections of spinal dorsal horn in normal rats (A, E, and I), and rats treated with vehicle (DCC+vehicle; B, F, and J), connecting segment-1 (CS-1; DCC+CS-1; C, G, and K), or fibronectin (DCC+FN; D, H, and L), at 14 days after DCC. The sections were collected from the lesion epicenter (A, B, C, and D) both rostral (E, F, G, and H), and caudal (I, J, K, and L) to the injury site (scale bar=100 mm). Quantification of spinal tissue 5-HT levels in epicenters and at the areas both rostral and caudal to the injury site collected from rats treated with vehicle, CS-1, or fibronectin (n=3 each group) at 1 day (M), 14 days (N), and 8 months (O) after DCC, and age-matched normal rats (^xversus normal rats; *versus vehicle-treated DCC rats).

FIG. 5.

Fibronectin promotes plasticity of serotonin (5-HT) fibers in the dorsal white matter. After dorsal column crush (DCC) and vehicle treatment, 5-HT immunoreactivity was similar to that of controls, with a slight increase observed in the dorsal white matter in both the corticospinal tract (CST, top row; A, B, and C) and the dorsal column (DC, bottom row; E, F, and G), at 1 day (A and E), 14 days (B and F), and 8 months (C and G) after DCC. However, fibronectin treatment induced abundant sprouting of descending 5-HT fibers with numerous intensely-stained fibers apparent in the dorsal white matter (n=3 each group; ^xversus normal rats; *versus vehicle-treated DCC rats). (D) The lesion in the spinal cord caused by DCC is indicated by the dashed line. The boxed areas denote regions selected for quantification of 5-HT immunoreactivity in the CST (L-DC, left dorsal column; R-DC, right dorsal column). (H) A representative 5-HT-immunostaining image taken from the L-DC, R-DC, and CST.

FIG. 6.

The anti-allodynia effects of fibronectin are blocked by both para-chlorophenylalanine (p-CPA) and methysergide. Effects of p-CPA and methysergide on mechanical Allodynia were evaluated following treatment with vehicle, connecting segment-1 (CS-1), or fibronectin (FN), 2 or 5 weeks post-DCC. CS-1, fibronectin, or vehicle was administered immediately after DCC. p-CPA and methysergide were then administered at 2 or 5 weeks post-DCC, and mechanical allodynia was assessed (n=3 per group; *versus rats with no antagonist treatment).