Cerium Nitrate Treatment Provides Eschar Stabilization through Reduction in Bioburden, DAMPs, and Inflammatory Cytokines in a Rat Scald Burn Model

Abstract

In this study, we used a clinically relevant rat scald burn model to determine the treatment effects of cerium nitrate (CN) for stabilizing burn eschars through reduction of damage-associated molecular patterns (DAMPs), inflammatory cytokines, and bioburden. Forty-two male Sprague-Dawley rats were anesthetized before undergoing a scald burn at 99°C for 6 seconds to create a 10% full-thickness burn. The test groups included sham burn, burn with water bathing, and burn with CN bathing. End point parameters included circulating DAMPs, proinflammatory cytokines, tissue myeloperoxidase activity, and quantification of resident flora in burn skin. The high mobility group protein box 1 was found to be elevated in burn animals at postoperative days (POD) 1 and 7. CN significantly alleviated the increase (P < .05 at POD 1 and P < .01 at POD 7). CN also lessened the heightened levels of hyaluronan in burn animals (P < .05 at POD 7). Additionally, CN significantly reduced the burn-induced increases in interleukin-1β, growth-regulated oncogene/keratinocyte chemoattractant, and macrophage inflammatory protein-1α in burn wounds. The anti-inflammatory effect of CN was also demonstrated in its ability to mitigate the upregulated circulatory xanthine oxidase/dehydrogenase and increased tissue neutrophil infiltration in burn animals. Last, CN suppressed postburn proliferation of resident skin microbes, resulting in a significant 2-log reduction by POD 7. In conclusion, these results suggest that CN attenuates the burn-induced DAMPs, tissue inflammatory responses, and regrowth of resident skin flora, all of which collectively could improve the quality of burn eschar when applied at the point of injury in prolonged field care situations.

Figure 1.

(A) Schematic illustration of burn and treatment approaches and end point procedures. Sham burn group consisted of three rats each postoperative days (POD), and burn + water and burn + cerium nitrate (CN) consisted of nine rats each POD. (B) Implementation of rat bathing system. A plastic square weigh boat (12.5 × 12.5 × 2 cm) was used as the basin for delivering the CN treatment (i.e., bathing) to the test animals. The prewarmed bathing solution was kept at 37°C with the heating block set at 40°C.

Figure 2.

Representative image of full-thickness burn wounds for comparison of burns with or without cerium nitrate (CN) treatment. Note the yellowish discoloration of burn wound skin in CN-treated burns at postoperative days (POD) 1 and 7. Also note that the burn wounds were not desiccated enough to form the typical firm eschar crust reportedly observed in CN-treated burns.

Figure 3.

Cerium nitrate (CN) reduces circulatory damage-associated molecular pattern (DAMP) levels in burn animals. Serum samples were collected from sham and burn rats with or without CN and assessed for DAMPs listed. Data are expressed as the mean ± SEM for three to six rats per group. *P < .05 burn + CN vs. burn + water; two-way ANOVA with multiple comparisons.

Figure 4.

Burn induces upregulation of proinflammatory cytokine profiles and cerium nitrate (CN) can mitigate the heightened level of cytokines in burn tissues. The supernatants from homogenized full-thickness (FT) burn skin were collected and assessed by a multiplex cytokine profile panel. Tissue levels of cytokines are expressed as the mean ± SEM for three to six rats per group (****P < .0001, **P < .01, *P < .05 burn + CN vs. respective sham burn or burn + water; two-way ANOVA with multiple comparisons).

Figure 5.

Cerium nitrate (CN) treatment retards the burn-induced local and systemic inflammation. (A) The supernatants from homogenized burn skin were collected and assessed for myeloperoxidase (MPO) activity. MPO data are expressed as the mean ± SEM for three to nine rats per group (*P < .05 burn + water vs. sham burn; two-way ANOVA). (B) Serum samples were collected from sham and burn rats and assessed for xanthine oxidase/xanthine dehydrogenase (XO/XDH) by ELISA. The data are expressed as the mean ± SEM for three to six rats per group (***P < .001 burn + CN vs. burn + water; two-way ANOVA with multiple comparisons).

Figure 6.

Cerium nitrate (CN) inhibits the proliferation and colonization of resident skin flora in burn skin. (A) Total bacterial CFU recovered from burn tissue at the indicated postoperative days (POD) was determined by serial dilution and plating on regular Trypticase soy blood agar. The data were transformed to log scale and then expressed as the mean ± SEM for three to nine rats per group (****P < .0001 burn + water vs. sham burn group; **P < .01 burn + CN vs. burn + water group; two-way ANOVA with multiple comparisons). (B) Total bacterial numbers were also determined by universal bacterial primers. The data were transformed to log scale and then expressed as the mean ± SEM for three to nine rats per group (****P < .0001 burn + water vs. sham burn group and burn + CN vs. burn + water group, respectively).