Resolvin D2 Limits Secondary Tissue Necrosis After Burn Wounds in Rats

Abstract

Secondary burn necrosis is the expansion and deepening of the original burn injury several days after injury. Limiting the extent of secondary burn necrosis may improve outcomes. In this study, we examined the ability of the lipid mediator of inflammation-resolution resolvin D2 (RvD2) and chromatin-lysing enzyme (DNase) to reduce secondary burn necrosis. Male Wistar rats were injured using a brass comb with 4 prongs heated in boiling water. This method created 2 parallel rows of 4 rectangular burned areas separated by 3 unburned interspaces. Starting at 2 hours after the burn injury, rats received either 25 ng/kg RvD2 intravenously daily for 7 days or 200 U/kg DNase every 12 hours for 3 days. We documented the necrosis around the initial wounds by digital photography. We used laser Doppler to assess the total blood flux in the burn area. We evaluated the functionality of the capillary network in the interspaces by optical coherence tomography angiography. We performed histological examination of wound skin tissue samples collected at 14 days postburn. We found that the interspace areas were preserved and had higher blood flow in the RvD2-treated group, while the burn areas expanded into the interspace areas, which were confluent by 7 days postburn, in the control-untreated group. We found a larger monocyte-to-neutrophil ratio in the RvD2-treated group compared with the DNase-treated and control groups (P < .05). Overall, RvD2 suppresses secondary necrosis and starts regeneration, highlighting the role of inflammation resolution as a potential therapeutic target in burn care.

Secondary necrosis of tissues surrounding burns of any size is a process that increases the size and depth of burn wounds. Through secondary necrosis, a small partial-thickness burn seen early after injury can progress to a larger full-thickness burn during the ensuing 24 to 48 hours. Three zones are described in the area burn wounds: a zone of necrosis at the center of the wound, which is surrounded by a zone of stasis and a zone of hyperemia at the edges.¹

Secondary necrosis has several consequences, which include the need for repeated surgical excisions to remove all nonviable tissue before wounds can be covered with split-thickness skin autografts.² Long-term consequences of repeated surgeries include deforming contractures, unstable scars, suboptimal function, and poor aesthetics. Moreover, full-thickness burns have also been associated with impairment of the immune, metabolic, and cardiovascular systems.³ Thus, preservation of the zone of stasis is important for limiting the secondary tissue necrosis seen in burn wound conversions. However, no current treatments reliably limit this process,^4–6 reflecting the poor understanding of the mechanisms behind secondary necrosis. Two major mechanisms for secondary necrosis around burn wounds have been proposed in the literature, including the loss of blood flow to the dermis surrounding the burn area⁷ and local inflammation due to the activation of neutrophils and other immune cells by the damaged tissue.⁸

One emerging paradigm for the termination of vascular patency is thrombosis triggered by the interaction of platelets with circulating chromatin.⁹ Circulating levels of extracellular chromatin have been measured to increase 10-fold in burn patients compared with healthy controls during the first week in the hospital and are correlated with the severity of injury^10–13 and with increased morbidity and mortality.¹⁴ One important source of chromatin in the circulation is presumably from neutrophils via the release of “neutrophil extracellular traps” (NETs). NETs are likely to be released by neutrophils activated immediately after burns^15,16 and the production of chromatin precipitates both microvascular¹⁷ and macrovascular thrombosis⁹ delaying wound healing. The enzyme DNase degrades NETs, and reverses these wound effects, and may play a role in burn wound necrosis through its actions upon chromatin in the circulation.⁹

The roles of inflammation in secondary wound necrosis and in particular the alterations in the balance between the inflammatory neutrophils and regenerative macrophages/monocytes after burns is under increasing scrutiny.^18,19 The lipid mediators of inflammation resolution are of great interest in restoring the inflammatory homeostasis for a broad range of other inflammatory diseases.²⁰ Some of these mediators like the resolvin D2 (RvD2) have already been shown to be of benefit in animal models of burn injury^8,21,22 and sepsis.^21,23 Intravenous (IV) administration of nanogram amounts of RvD2 up to 4 hours after burn injury in mice reduced secondary burn wound necrosis by preventing capillary thrombosis and neutrophil-induced tissue damage. Furthermore, resolvins may play a role in limiting the postburn systemic inflammatory response, with potentially protective effects on end-organ perfusion. Daily injections of RvD2 for 7 days after burn injury in a rat model prevented secondary kidney injury, liver damage, and death after endotoxin shock²² and cecal-ligation–induced peritonitis.²¹ A better understanding of how RvD2 accomplishes its beneficial actions is critical for developing new therapies.

The purpose of this study was to investigate further whether RvD2 and DNase improve burn wound healing. The goal was to evaluate the mechanisms responsible for secondary necrosis of burn wounds and to compare the efficacy of these 2 potential therapies. We measured the evolution of burn wounds in a comb-burn rat model, compared changes in blood flux using laser Doppler (LD), capillary perfusion by optical coherence tomography (OCT) modalities,²⁴ and evaluated skin histology in response to administration of RvD2 and DNase and their ability to limit secondary necrosis of the burn wound.

MATERIALS AND METHODS

Comb Burn Injury Rat Model

Male Wistar rats, weighing 320–420 g (Charles River Laboratories, Wilmington, MA) were used in experiments, after a 4- to 7-day acclimatization period. We removed the animal’s dorsal hair by shaving and with a depilatory cream (Magic Razorless Extra Strength Shave Cream for Men, SoftSheen Carson Laboratories, Westfield NJ) twice, at 24 hours and just before burn injury. After general anesthesia, we marked the areas of burn injury by 2 dotted lines, along the dorsal spine and a horizontal line along the forepaws. We then employed a 4-pronged rectangular metal comb to make 2 sets of burn wounds, 1 on each side of the back (1 cm lateral to the dotted line along the dorsal spine and 1 cm caudal from dotted line along the forepaws). The brass metal comb had 4 pins, between 8 and 10 mm wide, 18.5 mm long, and separated by 5 mm spaces (see Figure 1, Supplemental Digital Content, http://links.lww.com/BCR/A28). We immersed the metal comb in boiling water for at least 10 minutes before the injury. For each burn injury, we applied the hot comb to the back of the rats, for 20 seconds. We controlled the force pushing the comb, to an estimated 1 N force, the equivalent of 100 g weight pressing the comb against the skin. This procedure resulted in full-thickness burns. All rats were kept on a thermostat water blanket until recovery from anesthesia, when they were moved into individual cages. All procedures were conducted using aseptic techniques, and all animal protocols were approved by the Institutional Animal Care and Use Committee of Massachusetts General Hospital. Experiments were performed on 3 groups of rats: control (untreated), RvD2-treated, and DNase-treated.

Administration of RvD2 and DNase Treatment

RvD2-Treated Group.

Starting at 2 hours after the burn procedure, RvD2 (Cayman Chemical, Ann Arbor, MI) was administered daily via IV tail veil injections for 8 days, as previously described.²¹ Early experiments looked at RvD2 treatment with 2 doses of RvD2, either 25 ng/kg or 100 ng/kg of RvD2 in 0.3% ethanol (Supplementary Figure 1B). With the early data, we immediately modified our experiments to using only the lower dose, 25 ng/kg, treatment of RvD2.

DNase-Treated Group.

Starting at 2 hours after the burn procedure, 200 U/kg of Turbo DNase (Life Technologies, Grand Island, NY) was administered by IV and by intraperitoneal injections (67 U/kg IV and 133 U/kg intraperitoneal). Thereafter, DNase was administered every 12 hours, for 2 days for a total of 5 doses.

Control Group.

Control rats received a similar volume of vehicle using 0.3% ethanol, at the same time points as that of the RvD2 group.

Images and Measurements of Burn Wound and Interspace Area

Photographs were taken of the burn wounds at several time points: immediately after burn injury at day 0 and at days 1, 2, 3, 5, 7, and 14 days postburn (dpb). A ruler was included with each image to allow for calibration of measurements. Image J software National Institutes of Health (Washington, DC) was used to evaluate the wound and interspace areas. Two trained observers independently analyzed each image and manually traced the edge of the area of necrosis surrounding the burn. The total wound area was assessed for each animal; results were normalized to the size of wounds at day zero. The width of normal tissue separating each of the 4 comb wounds was measured and normalized to the width at day zero (interspace area).

LD Images for Blood Flux Assessment

We employed a LD line scanner (Moor LDLS-BI, Moor Instruments Inc., Wilmington, DE) to evaluate blood perfusion in the tissue surrounding the burn wound. We performed measurements before the burn, at 2 hours postburn, and at 1, 2, 3, 5, 7, and 14 dpb. Rats were anesthetized with 3% isoflurane. We quantified the blood flux in the interspace areas and on the sides of the wounds (defined the area of 5 mm distance from the edge of wound). Motion artifact secondary to respiration was removed and blood flux was measured in corresponding areas between the Doppler image and an automatic synchronized picture. Areas with blood perfusion above 600 PU threshold were classified as hyperemia.

OCT Angiography for Capillary Perfusion Measurements

Rats were anesthetized with inhalant of 3% isoflurane, and the duration of anesthesia for the imaging procedure was below 20 minutes. Following satisfactory anesthesia, rats were placed in lateral position on a polycarbonate stage. Imaging with OCT consisted in sending a low-power infrared light beam through the optical glass window and measuring the backscattered light.^17,18 Each imaging session was completed in approximately 20 minutes. After imaging, rats were placed back in their cages until fully recovered.

Quantification of Macrophages and Neutrophils in the Wounded Skin

To examine the histology of wound healing after burn injury, skin tissue specimens that included both burn eschar and healed area were harvested at 14 dpb and fixed in 10% phosphate-buffered formalin. Paraffin-embedded tissues were sliced into 4 µm sections and stained with H&E. Immunohistochemical staining for macrophages and neutrophils in the skin tissue samples was performed by HistoTox labs (Boulder, CO). To identify macrophages, anti-CD68 antibodies (Catalogue Number MCA341R, AbD Serotec, Raleigh, NC) were used at 1:2000 dilution and Proteinase K enzyme retrieval. To identify neutrophils, anti-myeloperoxidase antibodies (Catalogue Number PB9057, BosterBio, Pleasanton, CA) was employed at 2 ug/mL concentration in pH 6 citrate buffer, followed by heat-induced antigen retrieval. Slides and digitally scanned images of the tissue samples (pathxL 5.6.3, Belfast, United Kingdom) were provided for analysis. The total number of macrophages and neutrophils per field at 20× magnification for 3 representative fields were manually quantified in the dermis by a board-certified dermatopathologist on the basis of digital images (NEC AS241W 24” monitor, 1920 × 1080 resolution, Itasca, IL). Cell counts were analyzed for healthy dermis (defined as the area of viable dermis with corresponding intact epidermis and adnexal structures), burn (defined as the area of injured dermis with corresponding epidermal ulceration, necrosis, and fibrinoid scale crust), and interspace (defined as the region intervening 2 burn areas).

Statistical Analysis

Experimental data were expressed as mean ± SEM. The number of animals in each experiment is described in figure captions. Statistical tests: one-way analysis of variance, two-way analysis of variance with repeated measure and multiple comparison test where employed where appropriate. Differences were classified as significant at P values less than or equal to .05.

RESULTS

RvD2 and DNase Treatment Suppressed Wound Expansion

We induced burn wounds using a heated 4-pronged brass metal comb and monitored the progression of areas over 14 days (Supplementary Figure 1A). In the untreated control animals, the 4 wounds became confluent by 7 dpb and the average interspace area decreased by ~50% (N = 10; P ≤ 0.05; Figure 1A and [see Video, Supplemental Digital Content, http://links.lww.com/BCR/A102). In RvD2-treated rats, the interspace areas between the burn wounds remained intact, and the average interspace area decreased by ~20% by day 14 (N = 30 interspace areas; N = 5 rats). In the DNase-treated group, we observed a decrease of interspace area comparable with that in the control group up to 5 dpb and measured significant differences in the interspace area after 7 dpb (N = 30 interspace areas; N = 5 rats; P ≤ .05). Unlike the interspace area changes, the changes in total wound areas at 14 dpb were statistically similar between treated and untreated groups (N = 10 wounds; N = 5 rats), supporting the adequacy of the comb burn model for assessing the changes in the progression of burn areas with higher precision than the direct measurement of burn areas alone.

Figure 1.

Quantification of interspace and wound areas over time. A. Photographs were taken at 2 hours after burn injury (0 dpb) and repeated at 1, 2, 3, 5, 7, and 14 dpb. Interspace area is preserved in RvD2-treated animals and partially preserved in DNase-treated animals at 7 dpb. B. In the control group, the interspace area was reduced to 50% at 3 dpb when compared with 0 dpb. RvD2 treatment preserved the interspace from wound expansion. DNase treatment partially preserved the interspace at day 7. (N = 30 interspace areas, N = 5 rats for each group, control vs RvD2; *P ≤ .05, control vs DNase; ^ψP ≤ .05). C. Wound size area is not significantly different between treated and control groups (N = 10 wounds; N = 5 rats for each of the treated and control groups). dpb, days postburn; RvD2, resolvin D2.

RvD2 and DNase Promoted Blood Flow Around Burn Wound

To evaluate perfusion in the interspace area between the 3 groups, we employed a line scan LD. In conjunction with the morphological aspect of the wounds, this method enabled us to quantify the size of 3 typical zones at the wound sites: necrosis, hyperemia, and normal site¹ relative to the initial area of the wound (Figure 2A–C). We found that the size of the zone of necrosis was significantly smaller at 1 dpb in RvD2-treated rats (5 ± 5%) compared with control (40 ± 5%) and DNase-treated groups (30 ± 7%). The zone of necrosis remained significantly smaller in the RvD2-treated group at 3 and 5 dpb. The size of the zone of hyperemia increased from 1 to 5 dpb and was significantly larger in the RvD2-treated group compared with the untreated control. The size of the zone of hyperemia was also significantly larger at 3 dpb in the DNase-treated group compared with untreated control. The measured blood flow at the side of the wound was comparable for the 2 treatment groups and significantly higher than the untreated control for every day after 3 dpb (Figure 2D: N = 10 wounds; P ≤ .05).

Figure 2.

Quantification of the areas of necrosis, hyperemia, and stasis after burn injury and laser Doppler images. A–C. We evaluated the zone of necrosis by photographic images and the zone of hyperemia were based on laser Doppler images. The areas of hyperemia are significantly larger in RvD2- and DNase-treated groups at 3 and 5 dpb (N = 30 interspace areas, N = 5 rats for each group, control vs RvD2, DNase; ^ψp ≤ .05). RvD2 treatment could preserve the interspace at zone of normal site from wound expansion at 1 dpb (N = 30 interspace areas, N = 5 rats for each group, control vs RvD2; *P ≤ .05). D. The blood flow at the side of the wound was significantly higher in RvD2 and DNase treated groups than control group (N=10 wounds, N=5 rats for each group, Control vs RvD2, DNase; *p ≤ 0.05). RvD2, resolvin D2; dpb, days postburn.

Capillary Blood Flow at the Edge of Wounds Was Higher in the RvD2-Treated Group

Microvascular perfusion within the interspaces of the burn wound was further evaluated using OCT angiography. Both RvD2 and DNase-treated rats had increased blood flow (yellow areas) at the interface between the necrotic (no flow) and interspaces when compared with control groups at 3 dpb (Figure 3A). We calculated a perfusion index by locally averaging the OCT angiography signals in center and border area and comparing their intensities. We found a significant increase in the perfusion index in blood flow at the center of interspace area in the RvD2 treatment groups compared with control (25 ± 4% increase; N = 6 interspace areas; P ≤ 0.01). We also measured a significant increase in the perfusion index at the periphery of the interspace after DNase treatment compared with untreated control (30 ± 11%; N = 6 interspace areas; P ≤ .001; Figure 3B).

Figure 3.

Angiographic OCT images. A. We evaluated the flux of red blood cells at interspace area by OCT angiographic images at day 3 postburn. Resolvin D2 (RvD2) and DNase treatment revealed larger blood flow in the areas close to the wounds than control group. B. Quantification of blood flow shows significant increases in central blood flow RvD2-treated animals and border flow in DNase-treated animals (N = 6 interspace areas; N = 3 rats for each group, control vs RvD2, DNase; *P ≤ .05). Measurements were performed at 350 µm depth. OCT, optical coherence tomography. RvD2, resolvin D2.

More Macrophages Infiltrated the Burn Area at Day 14 in the Treated Groups

Histological changes in the skin tissue samples from control and treated animals were consistent with burn injury (see Figure 2, Supplemental Digital Content, http://links.lww.com/BCR/A101). In the burn wounds, we counted significantly more CD68-positive macrophages in the RvD2-treated groups (177 ± 22 cells/field of view; N = 15 fields; N = 5 rats per group; P ≤ .05) compared with DNase-treated (133 ± 16 cells/field) and untreated control samples (101 ± 14 cells/field, Figure 4). In the interspace area, the number of macrophages were lower in the RvD2-treated groups (29 ± 3 cells/field; N = 15 fields; P ≤ .05) and DNase-treated groups (43 ± 4 cells/field), compared with the untreated control (57 ± 4 cells/field). In the healthy tissue distant from the wound, the number of macrophages was comparable for all groups (8 ± 1, 11 ± 1, and 12 ± 3 cells/field for RvD2, DNase, and untreated control groups, respectively; N = 15 fields; P > .05). The interspace region was more difficult to discern in control compared with treatment groups, consistent with the macroscopic observations and measured differences in blood flow.

Figure 4.

Immunohistochemistry of interspace tissue, stained with anti-CD68 antibodies. Histopathologic images at 14 dpb in control, DNase, and RvD2 showing a representative microscopic field area for burn, interspace, and distant to demonstrate the relative number of CD68-positive macrophages identified per field (brown staining indicates positive immunoreactivity for CD68, 20× magnification). dpb, days postburn.

Fewer Neutrophils Infiltrated the Burn Area at Day 14 in the Treated Groups

The number of myeloperoxidase-positive neutrophils at all locations in the burn wounds was significantly lower in RvD2-treated groups compared with both DNase and untreated groups (Figure 5). The number of neutrophils in the burn area (22 ± 3, 30 ± 5, and 36 ± 4 cells/field for RvD2, DNase, and untreated control groups, respectively; N = 15 fields; N = 5 rats) was higher than in the interspace area (8 ± 1, 12 ± 2, and 14 ± 2 cells/field for RvD2, DNase, and untreated control groups, respectively) which in turn was higher than in the distant tissue (0.5 ± 0.1, 0.7 ± 0.3, and 0.9 ± 0.2 cells/field for RvD2, DNase, and untreated control groups, respectively; Figure 6).

Figure 5.

Immunohistochemistry of interspaced tissue stained with anti-MPO antibodies: histopathologic images at 14 dpb in control, DNase, and RvD2 showing a representative microscopic field area for burn, interspace, and distant to demonstrate the relative number of MPO-positive neutrophils identified per field (brown staining indicates positive immunoreactivity for MPO, 20× magnification). dpb, days postburn; MPO, myeloperoxidase.

Figure 6.

Quantitative analysis of immunohistochemistry images: the number of macrophages (CD68+) is highest and neutrophils (MPO+) is the lowest in RvD2-treated wounds at the burn site as compared with interspace and distant. The larger macrophage to neutrophil ratio suggests robust regeneration processes (N = 15 averages obtained for 3 representative 20× fields for each, N = 5 rats for each group, control vs RvD2; *P ≤ .05). MPO, myeloperoxidase; RvD2, resolvin D2.

DISCUSSION

In this study, we show that RvD2 and DNase are effective treatments that could prevent secondary burn wound necrosis. Both treatments are equally effective interventions for restoring blood flow to the interspace of comb-burn wounds. In addition, we observed that RvD2-treated rats had 2 times more macrophages infiltration into the dermis below the burned tissue when compared with both the DNase and control groups. The macrophage to neutrophil ratio at the burn site, following RvD2 treatment, was nearly double that of DNase treatment, both of which were higher than the ratio in controls. This finding is consistent with previous studies that found that the balance between the inflammatory neutrophils and regenerative macrophages/monocytes is dynamic¹⁹ and may be key to reducing secondary wound necrosis.¹⁸ In this study, the macrophage to neutrophil ratio appears to be more closely linked with reduced tissue necrosis, as opposed to extent of the inflammatory infiltrate alone. Together, these numbers suggest that after the administration of RvD2, the balance between tissue regeneration (driven by macrophages) and inflammation (driven by neutrophils) favors regeneration. These specific changes only occurred in the RvD2 treatment group.

The increase in the number of macrophages in the dermis suggests that regenerative processes have started around the wound area. Several findings from other studies support this claim: macrophages are recruited during the inflammatory phase of tissue formation and control vascular stability and the transition of granulation tissue into scar tissue.²⁵ The number of macrophages per skin surface unit after burn wounds increases by almost 300%.²⁶ In vivo, intradermal injection of human-activated macrophages at sulfur mustard–induced burn wounds decreased wound area.²⁷ Corroborated with our findings of improved blood flow, and suppressed wound expansion, the increased macrophage density suggests that RvD2 beneficial effects on burn wounds stem from reducing excessive inflammation.

The decrease in the number of neutrophils infiltrating the tissues surrounding the injury is key to preventing secondary necrosis. Although neutrophils play an essential role in controlling infection immediately postinjury, persistent neutrophil infiltration is reported to contribute to the chronic wound phenotype via reactive oxygen species–related tissue damage.^22,24 Clean, surgical wounds usually have the lowest amount of inflammation and heal the fastest.²⁸ Moreover, decreased neutrophil infiltration in the dermis has been correlated with more rapid healing and reduced scarring in porcine burn wounds,²⁹ while neutrophil depletion in mice following injection with rabbit anti-mouse neutrophil serum significantly accelerated wound reepithelialization.¹⁸

In conclusion, we showed increased blood flow and reduced proinflammatory infiltrate in the dermis after RvD2 and DNase treatment. RvD2 appears to also have potent selective actions in reducing excessive neutrophil trafficking to inflammatory loci. DNase appears to help mainly by maintaining capillary patency at the edge of the wound. While both processes appear to reduce the secondary necrosis around burn wounds, further studies will help reconcile the inflammatory and vascular paradigms of secondary necrosis, toward improved treatments to jump-start the regeneration processes and promote wound healing.

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This study was funded in part by grants from the National Institutes of Health (NIH - GM092804) and Shriners Burns Hospitals. Optical coherence tomography imaging was performed at the Center for Biomedical OCT Research and Translation, supported by NIH Grant Number EB015903.