Significance of alpha smooth muscle actin expression in traumatic painful neuromas: a pilot study in rats

Abstract

Treatment of painful neuromas remains a challenge and the mechanism of neuroma-associated pain is not yet fully understood. In this study, we aimed to observe the expression of alpha smooth muscle actin (α-SMA) in traumatic neuromas and to investigate its possible roles in the cause of neuropathic pain in a rat model. The rat sciatic nerve was used and the experiment was divided into two parts. In part I, our results showed significantly higher levels of α-SMA and the pain marker c-fos in the autotomy group than in the no-autotomy group. In part II, the expression of α-SMA in neuromas was down- and up-regulated using SB-431542 and GW9662, respectively. A significant correlation between autotomy scores and the expression level of α-SMA was found (R = 0.957; p < 0.001) and the expression level of α-SMA was positively related to the autotomy scores (R(2) = 0.915, p < 0.001). We concluded that the expression of α-SMA plays certain roles in the neuroma-associated pain, either as a direct cause of pain or as an indirect marker of existence of local mechanical stimuli. Our findings may provide new insights into the development of new treatment modalities for the management of intractable painful neuromas.

Figure 1. Increased level of α-SMA in the neuromas in the autotomy group.

(A) Immunohistochemistry analysis of α-SMA expression in the two groups at 4 weeks after transection (original magnification × 400). In the no-autotomy group, only slightly positive expression of α-SMA was seen; while in the autotomy group, extensively positive expression of α-SMA was present. The curved arrow shows the highly stained blood vessels and the straight arrow shows one of the positively stained myofibroblasts. (B) Protein expressions of α-SMA in the neuromas. GAPDH was used as the loading control and for band density normalization. (C) The optical density analysis of α-SMA proteins. Values are expressed as the mean ± SEM, n = 6 per group. *P < 0.05 versus the no-autotomy group.

Figure 2. Relative expression of c-fos in the dorsal horn of L4 spinal cord.

(A) Protein expression of c-fos of rats in no-autotomy group, autotomy group respectively. GAPDH was used as the loading control and for band density normalization. (B) The optical density analysis of c-fos in two groups. Values are expressed as the mean ± SEM, n = 6 per group. *P < 0.05 versus the no-autotomy group.

Figure 3. Results of weekly average autotomy scores.

Significant differences in the average autotomy score were observed among the three groups two weeks after surgery (*p < 0.05) except at the end of the first week (^▲p > 0.05: SB-431542 vs. control group, p = 0.818; GW9662 vs. control group, p = 0.645; SB-431542 vs. GW9662, p = 0.490) and between GW9662 group and control group at 2 weeks (^♦p = 0.25).

Figure 4. Effects of SB-431542 and GW9662 on the expression of α-SMA in the neuromas.

(A) Protein expression of α-SMA in control, SB-431542 and GW9662 groups, respectively. GAPDH was used as the loading control and for band density normalization. (B) The optical density analysis of α-SMA protein in three groups. Values are expressed as the mean ± SEM, n = 6 per group *P < 0.05 versus the control group, ^#P < 0.05 versus the control group.

Figure 5. Correlation between autotomy scores and the expression levels of α-SMA.

The scatter diagram demonstrated a positive correlation between the autotomy scores and expression levels of α-SMA (R² = 0.915, p < 0.001).

Figure 6. Effects of SB-431542 and GW9662 on the expression of c-fos in the dorsal horn of L4 spinal cord.

(A) c-fos expression in each group as assessed by immunohistochemistry (original magnification × 400). Significantly positive staining of c-fos in the neuron cells was seen in GW9662 group; in contrast, slightly positive staining of c-fos in the neuron cells was observed in the control group; while only very few neuron cells had positive staining of c-fos in the SB-431542 group. The arrows showed the neuron cells with positive staining of c-fos. (B) Protein expression of c-fos in control, SB-431542 and GW9662 groups, respectively. GAPDH was used as the loading control and for band density normalization. (C) The optical density analysis of c-fos protein in three groups. Values are expressed as the mean ± SEM, n = 6 per group *P < 0.05 versus the control group, ^#P < 0.05 versus the control group.