Vascular endothelial growth factor and spinal cord injury pain

Abstract

Vascular endothelial growth factor (VEGF)-A mRNA was previously identified as one of the significantly upregulated transcripts in spinal cord injured tissue from adult rats that developed allodynia. To characterize the role of VEGF-A in the development of pain in spinal cord injury (SCI), we analyzed mechanical allodynia in SCI rats that were treated with either vehicle, VEGF-A isoform 165 (VEGF(165)), or neutralizing VEGF(165)-specific antibody. We have observed that exogenous administration of VEGF(165) increased both the number of SCI rats that develop persistent mechanical allodynia, and the level of hypersensitivity to mechanical stimuli. Our analysis identified excessive and aberrant growth of myelinated axons in dorsal horns and dorsal columns of chronically injured spinal cords as possible mechanisms for both SCI pain and VEGF(165)-induced amplification of SCI pain, suggesting that elevated endogenous VEGF(165) may have a role in the development of allodynia after SCI. However, the neutralizing VEGF(165) antibody showed no effect on allodynia or axonal sprouting after SCI. It is possible that another endogenous VEGF isoform activates the same signaling pathway as the exogenously-administered 165 isoform and contributes to SCI pain. Our transcriptional analysis revealed that endogenous VEGF(188) is likely to be the isoform involved in the development of allodynia after SCI. To the best of our knowledge, this is the first study to suggest a possible link between VEGF, nonspecific sprouting of myelinated axons, and mechanical allodynia following SCI.

FIG. 1.

Categorization of uninjured and injured rats based on the percentage of mechanical threshold changes seen in the forelimbs after spinal cord injury (SCI), or sham treatment, versus pre-SCI baseline values, using K-means clustering. The y axis shows the number of rats analyzed, and the x axis the percentage change in forelimb mechanical thresholds at 4 weeks compared to baseline values (before sham treatment or SCI, set to 100%). (A) Sham rats (n = 25) showed a mean decrease of −0.23 ± 37.8%, while contused rats (SCI; n = 59) showed a mean decrease of −47 ± 34.93%. (B) After introducing a 40% criterion, a group of SCI rats (33 out of 59) showed significantly increased sensitivity to mechanical stimuli compared to sham rats.

FIG. 2.

(A) Analysis of mechanical allodynia, as described in Figure 1 and in the methods section. (A) Incidence of pain. Animals that had increased sensitivity in both forelimbs at all time points tested were considered to demonstrate persistent pain. None of the sham animals had persistent pain, while 8% of vehicle-treated spinal cord injury (SCI) rats and 34% of VEGF₁₆₅-treated animals had persistent pain. The chi square test showed a statistically significant difference between VEGF₁₆₅-treated and vehicle-treated SCI rats (*p < 0.05). (B) Pain levels. Shown are the percentage decreases in mechanical thresholds seen at 4, 6, and 8 weeks post-SCI in rats that developed persistent allodynia. Although the percentage decrease is represented by negative numbers in Figure 1, here we present it as a positive number (on the y axis), so a higher percentage indicates increased sensitivity (e.g., lower thresholds to mechanical stimuli or increased pain levels in post-SCI rats compared to pre-injury baseline levels). (C) Basso, Beattie, and Bresnahan (BBB) scale scores (mean ± standard deviation) of all SCI rats used in this study showed no effect of VEGF₁₆₅ administration on motor recovery after SCI (VEGF, vascular endothelial growth factor).

FIG. 3.

(A) A representative example of calcitonin gene-related peptide (CGRP) immunolabeling in sham rats (A_I); vehicle-treated spinal cord injury (SCI) rats (A_II), and vascular endothelial growth factor (VEGF)₁₆₅-treated SCI rats (A_III), that were classified as SCI rats that developed pain (scale bar = 100 μm; T6 segment at 8 weeks post-injury). (B) Quantitative analysis of the intensity of CGRP labeling in two segments rostral and two segments caudal from the site of injury (T6–T10; n = 6 animals per group; values normalized to sham = 1).

FIG. 4.

(A) Neurofilament (NF)-H and NF-L mRNA were assessed in spinal cord injury (SCI) rats with and without spontaneous pain (n = 4 per group) using DNA microarrays (a method previously described in Nesic et al., 2005). mRNA levels of NF-H and NF-L were significantly increased (*p < 0.05) in five segments rostral from the site of injury. mRNA values were normalized to the levels of mRNAs in SCI rats without pain (set to = 1; mean ± standard deviation). Similarly significant increases in NF mRNAs were also found in five segments caudal from the site of injury (data not shown). (B) A representative example of SMI-31 labeling in sham rats (B_I), vehicle-treated SCI rats (B_II), and VEGF₁₆₅-treated SCI rats (B_III), that were classified as SCI rats that developed pain (scale bar = 500 μm; T6 segment at 8 weeks post-injury). Normal labeling for SMI-31 (Sham) was significantly lower in vehicle-treated and VEGF₁₆₅-treated SCI rats. However, VEGF₁₆₅-treated SCI rats had visibly more SMI-31-labeled axons in the dorsal columns (marked with arrows), and dorsal horns, than vehicle-treated SCI rats. (B_IV–B_VI) SMI-31-labeled thick myelinated axons are widely scattered in sham and vehicle-treated dorsal horns, but are visibly increased in the dorsal horns of VEGF₁₆₅-treated cords (scale bar = 200 μm). (C) Quantitative analysis of SMI-31 labeling in the dorsal columns of spinal cord segments around the site of injury (T6–T10) in three experimental groups (n = 6 animals per group). Sham animals had significantly more SMI-31 labeling compared to both vehicle-treated and VEGF₁₆₅-treated animals (average ± standard deviation; **p < 0.0001; values normalized to sham = 1). However, VEGF₁₆₅-treated SCI rats had significantly more SMI-31-labeled axons in the dorsal columns than vehicle-treated SCI rats (*p < 0.01). (D) Semi-quantitative analysis of SMI-31 labeling in the dorsal horns in the same sections used for the analysis shown in C. (average ± standard deviation; *p < 0.05; values normalized to sham = 1). SCI pain was associated with significant twofold increases in SM-31 labeling (p < 0.05) in the dorsal horns, while VEGF₁₆₅-induced SCI pain was associated with additional significant increases in SM-31 labeling (2.5-fold; p < 0.05; VEGF, vascular endothelial growth factor).

FIG. 4.

(A) Neurofilament (NF)-H and NF-L mRNA were assessed in spinal cord injury (SCI) rats with and without spontaneous pain (n = 4 per group) using DNA microarrays (a method previously described in Nesic et al., 2005). mRNA levels of NF-H and NF-L were significantly increased (*p < 0.05) in five segments rostral from the site of injury. mRNA values were normalized to the levels of mRNAs in SCI rats without pain (set to = 1; mean ± standard deviation). Similarly significant increases in NF mRNAs were also found in five segments caudal from the site of injury (data not shown). (B) A representative example of SMI-31 labeling in sham rats (B_I), vehicle-treated SCI rats (B_II), and VEGF₁₆₅-treated SCI rats (B_III), that were classified as SCI rats that developed pain (scale bar = 500 μm; T6 segment at 8 weeks post-injury). Normal labeling for SMI-31 (Sham) was significantly lower in vehicle-treated and VEGF₁₆₅-treated SCI rats. However, VEGF₁₆₅-treated SCI rats had visibly more SMI-31-labeled axons in the dorsal columns (marked with arrows), and dorsal horns, than vehicle-treated SCI rats. (B_IV–B_VI) SMI-31-labeled thick myelinated axons are widely scattered in sham and vehicle-treated dorsal horns, but are visibly increased in the dorsal horns of VEGF₁₆₅-treated cords (scale bar = 200 μm). (C) Quantitative analysis of SMI-31 labeling in the dorsal columns of spinal cord segments around the site of injury (T6–T10) in three experimental groups (n = 6 animals per group). Sham animals had significantly more SMI-31 labeling compared to both vehicle-treated and VEGF₁₆₅-treated animals (average ± standard deviation; **p < 0.0001; values normalized to sham = 1). However, VEGF₁₆₅-treated SCI rats had significantly more SMI-31-labeled axons in the dorsal columns than vehicle-treated SCI rats (*p < 0.01). (D) Semi-quantitative analysis of SMI-31 labeling in the dorsal horns in the same sections used for the analysis shown in C. (average ± standard deviation; *p < 0.05; values normalized to sham = 1). SCI pain was associated with significant twofold increases in SM-31 labeling (p < 0.05) in the dorsal horns, while VEGF₁₆₅-induced SCI pain was associated with additional significant increases in SM-31 labeling (2.5-fold; p < 0.05; VEGF, vascular endothelial growth factor).

FIG. 5.

(A) The incidence of chronic allodynia in spinal cord injury (SCI) rats was not affected by anti-VEGF₁₆₅ treatment. (B) VEGF₁₈₈ mRNA levels assessed in SCI rats with and without spontaneous pain (n = 4 per group; mean ± standard deviation) using DNA microarrays were significantly elevated in SCI rats with pain (*p < 0.05; as previously described by Nesic et al., ; VEGF, vascular endothelial growth factor).