Fat Grafting in Burn Scar Alleviates Neuropathic Pain via Anti-Inflammation Effect in Scar and Spinal Cord

Abstract

Burn-induced neuropathic pain is complex, and fat grafting has reportedly improved neuropathic pain. However, the mechanism of fat grafting in improving neuropathic pain is unclear. Previous investigations have found that neuroinflammation causes neuropathic pain, and anti-inflammatory targeting may provide potential therapeutic opportunities in neuropathic pain. We hypothesized that fat grafting in burn scars improves the neuropathic pain through anti-inflammation. Burn-induced scar pain was confirmed using a mechanical response test 4 weeks after burn injuries, and autologous fat grafting in the scar area was performed simultaneously. After 4 weeks, the animals were sacrificed, and specimens were collected for the inflammation test, including COX-2, iNOS, and nNOS in the injured skin and spinal cord dorsal horns through immunohistochemistry and Western assays. Furthermore, pro-inflammatory cytokines (IL-1 β and TNF-α) in the spinal cord were collected. Double immunofluorescent staining images for measuring p-IκB, p-NFκB, p-JNK, and TUNEL as well as Western blots of AKT, Bax/Bcl-2 for the inflammatory process, and apoptosis were analyzed. Fat grafting significantly reduced COX2, nNOS, and iNOS in the skin and spinal cord dorsal horns, as well as IL-1β and TNF-α, compared with the burn group. Moreover, regarding the anti-inflammatory effect, the apoptosis cells in the spinal cord significantly decreased after the fat grafting in the burn injury group. Fat grafting was effective in treating burn-induced neuropathic pain through the alleviation of neuroinflammation and ameliorated spinal neuronal apoptosis.

Fig 1. The flowchart of this study and the behavior tests from various groups.

(A) A flowchart of the study D (day) and W (week). (B) The response thresholds to the mechanical stimuli decreased strongly on the burn injury side hind paws in Group C and Group D, 1–4 weeks post burn injury. After the fat grafting, Group D thresholds significantly increased in the following weeks versus Group C. (C) The response thresholds to the radiant heat stimuli did not show significant differences between the groups. The sample size was n = 6 for each group. (Data are plotted as mean ± SEM; ***P < 0.001; **P < 0.01; *P < 0.05).

Fig 2. IHC staining images and protein expressions of COX-2, iNOS, and nNOS in the hind paw skin.

(A) The effect of fat grafts on the expression of the inflammatory proteins,COX-2, iNOS and nNOS in the hind paw skin induced by a third-degree burn. (B) Western blot analyses of COX-2, iNOS and nNOS in the hind paw skin at week-8 after the burn injury. (Above) Western blot images were illustrated, and β-actin was used as the internal control. (Bottom) Quantitative result analyses were demonstrated, and the sample size was n > 3 for each test. The expressions of COX-2, iNOS and nNOS were decreased significantly in the burn with fat graft (Group D) compared with the burn with saline injection (Group C). *P < 0.05.

Fig 3. Fat grafting inhibited the expressions of inflammatory proteins, COX-2, iNOS and nNOS, in the spinal cord dorsal horn, and reduced the secreted levels of pro-inflammatory cytokines, IL-1β and TNF-α.

(A) Western blot analyses of COX-2, iNOS and nNOS in the spinal cord dorsal horn at 8 weeks after the burn injury. β-actin was used as the internal control. The presentation format is similar to Fig 2. Protein expressions of COX-2, iNOS and nNOS were decreased significantly in the burn with fat graft group compared with the burn with saline injection group (Group D vs. Group C). (B) Fat grafts appreciably reduced the secretions of the pro-inflammatory cytokines, IL-1β and TNF-α, in the spinal cord dorsal horn. IL-1β and TNF-α assessment was conducted at least in triplicate, n > 3, *P < 0.05.

Fig 4. Fat grafts increased p-IκB, reduced the p-NFκB protein, and through the inhibition of p-JNK, attenuated apoptosis in the spinal cord dorsal horn.

(A) Double immunofluorescent staining was performed to measure p-IκB and p-NFκB. Few p-IκB proteins were expressed in Group C, but p-IκB were increased in Group D. The expression of p-NFκB was abolished and reduced by fat grafts in Group D versus Group C. (B) Double immunofluorescence images in p-JNK and TUNEL expression were elevated following an increase in the TUNEL-positive cells. In Group D, we found that the inhibition from fat graft of p-JNK attenuated apoptosis in the spinal cord dorsal horn.

Fig 5. Fat graft regulated p-AKT/AKT and Bax/Bcl-2 ratios in the spinal cord dorsal horn.

(A and B) Both expression ratios of p-AKT/AKT and Bax/Bcl-2 increased significantly in Group C versus Group D. These results indicated that fat grafting reduced cell death of neuron in the spinal cord (Data were presented with mean ± SEM, **P < 0.01; *P < 0.05).