Topical application of cerium nitrate prevents burn edema after burn plasma transfer

Microvascular Research 78 (2009) 425–431

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Topical application of cerium nitrate prevents burn edema after burn plasma transfer Thomas Kremer a,⁎, F. Hernekamp a, K. Riedel a, Ch. Peter b, M.M. Gebhardt c, G. Germann a, Ch. Heitmann a, Andreas Walther b a Department of Hand, Plastic and Reconstructive Surgery, - Burn Center - BG Trauma Center Ludwigshafen, Plastic- and Hand Surgery, University of Heidelberg, Ludwig Guttmann Str. 13, 67071 Ludwigshafen, Germany b Department of Anesthesiology, University of Heidelberg, Neuenheimer Feld 110, 69120 Heidelberg, Germany c Experimental Surgery, University of Heidelberg, Neuenheimer Feld 364, 69120 Heidelberg, Germany

a r t i c l e

i n f o

Article history: Received 20 February 2009 Revised 28 July 2009 Accepted 29 July 2009 Available online 4 August 2009 Keywords: Burn plasma transfer Burn shock Burn edema Capillary leakage Leukocyte activation Intravital microscopy Cerium nitrate Microcirculation Thermal injury

a b s t r a c t Thermal injuries of more than 20% body surface area (BSA) result in systemic capillary leakage with subsequent edema. This can similarly be induced by burn plasma transfer (BPT) from burned individuals to healthy rats. We evaluated if cerium nitrate (CN) bathing can prevent edema after BPT. Therefore, donor rats (DR) underwent thermal injury (100 °C water, 30%BSA, 12 s) for positive controls and were additionally bathed in CN (0.05M, at 10 and 120 min) for study groups. For negative controls DR underwent shamburn (37 °C water, 30%BSA, 12 s). DR-plasma (harvested 4 h post trauma) was transferred to healthy individuals. Intravital microscopy was performed in mesenteric venules (0/60/120 min). Edema was assessed by FITCalbumin extravasation. Additionally, leukocyte sticking (cells/mm2) and micro hemodynamic parameters were assessed. Significant systemic capillary leakage was observed after BPT at 120 min. Edema formation was significantly lower in negative controls. Topical CN application after 10 and 120 min reduced FITC-efflux to baseline levels. Adherent leukocytes increased slightly in all groups. Leukocyte-sticking tended to be reduced after CN bathing. In conclusion, BPT induces burn edema in healthy individuals. CN bathing after 10 and 120 min reduces mediator levels in burned individuals. Therefore, BPT after CN application does not induce burn shock anymore. Burn edema is partially independent from leukocyte activation because CN significantly influences macromolecular leakage whereas leukocyte activation is not significantly altered. © 2009 Elsevier Inc. All rights reserved.

Introduction Wound infections after major burn injuries are a major cause of death in burn victims. Consequently, the search for an ideal topical antiseptic agent with high antimicrobial efficacy and low toxicity was performed for decades. Cerium nitrate was used as a topical antiseptic agent for the treatment of burn wounds since the 1970s. It was found to be especially effective for gram negative bacteria and fungi (Monafo et al., 1978, 1976). In contrary, sulfadiazine silver solutions were found to predominantly reduce the gram positive flora. Therefore, cerium nitrate and sulfadiazine silver were combined. Monafo et al. found cerium nitrate alone and combined with sulfadiazine silver to reduce wound infections after burns (Monafo et al., 1976). Furthermore, cerium nitrate was found to reduce the number of anticipated deaths in burn patients (Monafo et al., 1978, 1976; Scheidegger et al., 1992). This reduction of burn related mortality cannot be explained by the control of wound infections alone, because a single treatment with cerium nitrate bathing on day one after burn injury is still effective,

⁎ Corresponding author. Fax: +49 621 6810 2609. E-mail address: [email protected] (T. Kremer). 0026-2862/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.mvr.2009.07.006

even if wound infections may occur later (Scheidegger et al., 1992). Furthermore, burn related mortality has been shown to be associated to the systemic inflammatory response syndrome rather than uncontrolled infection and related complications (Huang et al., 1998; Sheridan et al., 1998). The systemic inflammatory response with subsequent capillary leakage and burn edema has been shown to be at least partially induced by leukocyte activation and the consecutive release of several immunomodulative cytokines (Algöwer et al., 1995; Horton et al., 2004; Maass et al., 2002a,b; Sparkes, 1996). Schoenenberger et al. postulated immunosuppressive material – described as lipid protein complex (LPC) – in the burn eschar to cause leukocyte activation after burn injury (Schoenenberger et al., 1975). Furthermore, cerium nitrate was shown to directly bind LPC (Kremer et al., 1981). Consequently, Eski et al. were able to show that cerium nitrate bathing reduces rolling, sticking and transmigrating leukocytes after burn injury in an intravital microscopic study in the rat cremaster muscle (Eski et al., 2001). The same group reported that IL-6 and TNFalpha levels are similarly altered after early burn wound excision as well as cerium nitrate treatment (Deveci et al., 2000). However, the systemic inflammatory response is not only characterized by immunologic activation but by systemic capillary leakage, which occurs as early as 2 to 4 h post trauma (Cioffi, 2001; Gibran and Heimbach, 2000; Lund et al., 1992). Excessive loss of blood

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fluid and plasma proteins results in hypovolemic shock, respiratory distress syndrome and generalized malperfusion, subsequently leading to multiple organ failure (Arturson, 2000; Lund et al., 1992). Endothelial damage in the systemic inflammatory response to endotoxemia has been shown to be independent from leukocyte activation (Walther et al., 2003, 2000). Similarly, capillary leakage seems to be at least partially independent from leukocyte activation after burn injury (Kremer et al., 2008). Therefore, the aim of the present study was to evaluate if cerium nitrate treatment is effective in the reduction of leukocyte activation and systemic capillary leakage with subsequent systemic edema formation. We have been able to show, that the transfer of blood plasma (harvested as early as 4 h post trauma) from individuals after burn injury to healthy animals leads to systemic burn edema in a rat model. Furthermore, we observed leukocyte activation (indicated by leukocyte sticking) after burn plasma transfer. However, differences to negative and positive controls were not significant (Kremer et al., 2008). This model clearly proves that plasma factors rather than wound infection induce systemic burn shock after burn injury. Therefore, we choose this new model to evaluate if cerium nitrate treatment effectively reduces these plasma factors, which may be predominantly immunomodulative cytokines or LPC, to prevent systemic burn edema. Furthermore, the role of inhibition of leukocyte activation by cerium nitrate for systemic burn edema was elucidated. Materials and methods

and water ad libitum until the day before the experiment. Food was withheld from all animals for 4 h before the experiment; free access to water was maintained. Thermal injury was inflicted based on a modified Walker and Mason burn model (Huang et al., 2003; Korompai and Yuan, 2002; Kremer et al., 2008; Walker and Mason, 1968). A dorsal area that equals 30% TBSA was shaved. The rat was placed in a mold with an adjustable opening to expose the shaved area to 100 °C for 12 s. This produces a clearly defined full-thickness burn without detectable visceral injury as confirmed by pathological studies (Korompai and Yuan, 2002). For controls (sham burn; SB) rats underwent the same burn procedure except the water temperature was 37 °C. Donor rat procedure Donor rats were randomized in 4 groups. All donors were anesthesized intraperitoneally with 60 mg/kg body weight sodium pentobarbital (Nembutal; Sanofi, Düsseldorf, Germany) and intramuscular injection of Ketamine S. Donor-Group 1 (n = 4) underwent thermal injury as described and was bathed in saline 10 min after burn injury. Donor-Groups 2 and 3 (n = 4 each) underwent the same burn procedure but were bathed in cerium nitrate (0.05 M in saline for 5 min) after 10 min or 120 min, respectively. Donor-Group 4 animals (n = 3) underwent the sham burn procedure as described and were bathed in saline 10 min post trauma. All donor rats were sacrificed 4 h post burn and the blood plasma was harvested according to established protocols (Carlson et al., 2002; Huang et al., 2003).

Experimental protocol Study group animals All experimental procedures and protocols used in this investigation were reviewed and approved by the Governmental Animal Protection Committee. Male Wistar rats (250–350 g body weight) were maintained in an animal facility with a 12-h light–dark cycle and housed in stainless-steel cages in a temperature- and humiditycontrolled room. All animals were kept on a diet of standard rat food

Rats were randomized in 4 groups of recipient rats that received plasma from donor animals. Recipient rats were anasthesized as described for donors. In positive controls (n = 14; BP) a burn plasma infusion was performed in a 10% dilution in saline, which reliably induces systemic burn shock in the recipient rats (Kremer et al.,

Fig. 1. The experimental protocol: Positive controls (BP) underwent plasma infusion from donor rats after burn injury (Donor group 1), Study groups underwent plasma infusion of donor rats, which were treated with cerium nitrate bathing 10 min (CN10) and 2 h (CN120) post trauma (Donor groups 2 and 3). Negative controls received plasma from donor rats after sham burn (Donor group 4; SB). Intravital microscopy was performed at 0 min, 60 min and 120 min for leukocyte sticking and plasma extravasation of FITC-albumin.

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2008). Rats of the two study groups (CN10 [n = 14] and CN120 [n = 14]) underwent a plasma infusion of donor groups 2 and 3, which was similarly diluted to 10% in physiologic saline. Negative control rats (n = 11; SB) underwent a sham burn plasma infusion (10% in saline), which was harvested from group 4 donors. The amount of fluid was calculated according to the Parkland formula with a virtual burn of 30% TBSA (0.25 ml/kg/30%TBSA/h; Fig. 1). Animal preparation Under anesthesia (see above), the left carotid artery was cannulated with polyethylene tubing (outer diameter, 0.9 mm; inner diameter, 0.5 mm) for the measurement of mean arterial pressure via a pressure transducer connected to a monitoring system (Servomed, Hellige, Germany). For plasma infusion and fluid resuscitation, the right jugular vein was cannulated with the same tubing. Rectal temperature was measured with a thermistor probe and maintained at 37 °C with a heating lamp. The abdomen was opened via a midline incision and the ileal portion of the mesentery was carefully spread over a plastic stage. The preparation was continuously superfused with a thermostatcontrolled (36.5 °C), bicarbonate-buffered salt solution (132 mM of sodium chloride, 4.7 mM of potassium chloride, 2 mM of calcium chloride, 1.2 mM of magnesium chloride, and 18 mM of sodium bicarbonate) equilibrated with 5% CO2 in nitrogen to adjust the pH to 7.35 (average volume: 160 ml/animal). Animal preparation and mesenteric exterioration were followed by a 30 min stabilization period on the stage of the intravital microscope. All animals received PKH26-GL labeled erythrocytes and fluorescein isothiocyanate-labeled bovine albumin 15 min before the first measurement and the simultaneous onset of plasma infusions (BP, CN10, CN120 and SB; Fig. 1). Intravital microscopy The mesenteric microcirculation was observed using a specially designed microscope (Orthoplan, Leica, Wetzlar, Germany) equipped with a 40-fold objective (Achroplan 40/0.75 W; Zeiss, Jena, Germany). The exteriorized mesentery was visualized either by transillumination (150 W cold light fountain; KL 1500 electronic, Schott, Wiesbaden, Germany) or by epi-illumination using an epifluorescence illuminator (Ploemopak; Leica), which consisted of a 100 W short arc mercury lamp (Osram, Munich, Germany) and a filter system for the fluorescence excitation (green light excitation: N 2.1; blue light excitation: I 3; Leica). To protect the preparation from heat, a heat protection filter (KG1; Leica) was located in the body of the microscope. Microscopic

Fig. 2. Number of new adherent leukocytes (cells/mm2) after burn plasma infusion (BP), burn plasma infusion after additional treatment with cerium nitrate 10 min post trauma (CN10) and 2 h post trauma (CN120) and sham burn plasma infusion (SB). Differences were not significant between groups and within groups compared vs. baseline.

images were transferred to a monitor (PVM 1444QM; Sony, Tokyo, Japan) by a low light camera (Kappa CF 8/1; Kappa Messtechnik, Gleichen, Germany) and recorded on videotape using a video recorder (Panasonic S-VHS AG-7350-E; Matsushita, Japan). Measurement of erythrocyte velocity Mean red blood cell velocities (VRBC) in single unbranched postcapillary venules (20–35 μm diameter) were analyzed off-line at 0, 60, and 120 min after administration of burn or sham burn plasma with a computer-assisted microcirculation analysis system (Cap image; Zeintl, Heidelberg, Germany). Therefore, fluorescent-labeled erythrocytes from donor rats were injected before microscopy (0.5 ml/kg body weight; hematocrit, 50%). These erythrocytes were labeled with a red fluorescent cell linker kit (PKH26-GL; Sigma Chemical, Deisenhofen, Germany) using a modified procedure according to Horan et al. (1990). For velocity measurement, the distance through which a labeled erythrocyte traveled within two subsequent video frames was divided by the known video frame time interval of 20 ms. Mean blood cell velocity in a vessel was calculated using the harmonic mean velocities of 20 individual erythrocytes. Volumetric blood flow (Q˙v) was calculated from the product of VRBC and venular crosssectional area (πD2 / 4) assuming a cylindrical shape of the vessel. To calculate venular wall shear rate, the vessel diameters (Dv) of the same venules were measured (Cap image; Zeintl). Venular wall shear rate (γ) was calculated based on the Newtonian definition: γ = 8 (VRBC / Dv) (House and Lipowsky, 1987).

Table 1 Microhemodynamic alterations in postcapillary venules after burn plasma infusion (BP), burn plasma infusion after additional treatment with cerium nitrate 10 min post trauma (CN10) and 2 h post trauma (CN120) and sham burn plasma infusion (SB). Variable

Venular diameters [μm]

Volumetric blood flow [μm³/s]

Venular wall shear rate [1/s]

Erythrocyte velocity [mm/s]

Group

SB CN10 CN120 BP SB CN10 CN120 BP SB CN10 CN120 BP SB CN10 CN120 BP

Time during plasma infusion 0 min

60 min

120 min

25.4 ± 2.0 28.8 ± 1.3 28.2 ± 2.2 26.9 ± 1.8 .76 ± .12 .81 ± .18 1.26 ± .19 .95 ± .12 579.2 ± 74.2 344.9 ± 44.6 577.5 ± 132.1 554.2 ± 64.4 1.7 ± .2 1.2 ± .2 2.0 ± .2 1.8 ± .2

25.3 ± 1.6 29.0 ± 1.3 28.4 ± 2.8 27.7 ± 1.9 .62 ± .10 .87 ± .15 .95 ± .18 1.09 ± .26 428.6 ± 57.0 377.5 ± 56.7 419.0 ± 116.3 601.8 ± 118.6 1.3 ± .2 1.3 ± .2 1.5 ± .5 1.9 ± .3

25.1 ± 2.1 29.2 ± 1.3 29.3 ± 3.2 28.5 ± 1.9 .55 ± .12 .84 ± .16 .98 ± .06 .96 ± .23 369.2 ± 73.0 354.8 ± 44.1 414.8 ± 104.0 477.1 ± 49.9 1.1 ± .2 1.3 ± .2 1.5 ± .1 1.6 ± .2

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studied. New adherent leukocytes at 60 min and 120 min were defined as difference between leukocytes that were adherent to the endothelium at 60 min and 120 min and adherent leukocytes at baseline. Quantitation of macromolecular leakage

Fig. 3. Plasma extravasation of FITC-albumin (ratio of interstitial to venular fluorescence intensity [Ii/Iv]) after burn plasma infusion (BP), burn plasma infusion after additional treatment with cerium nitrate 10 min post trauma (CN10) and 2 h post trauma (CN120) and sham burn plasma infusion (SB). (#; significant differences vs. baseline, ⁎; significant differences vs. BP, $; significant differences vs. SB, CN10 and CN120. For detailed information see Table 2).

Leukocyte–endothelial interactions The behavior of leukocytes was evaluated in postcapillary venules of approximately the same diameter (20 to 35 μm; Table 1) and was visualized by using transillumination microscopy at 0, 60, and 120 min. Adherent leukocytes were determined off-line during playback of the videotaped images. Adherent leukocytes were defined as cells that do not move or detach from the endothelial wall for a period of N= 30 s. Leukocyte adherence was expressed as the number of cells per square millimeter of vessel surface as calculated from the diameter and length of the vessel segment

To quantify albumin leakage across mesenteric venules, 50 mg/ kg body weight of fluorescein isothiocyanate-labeled bovine albumin (Sigma Chemicals, diluted in saline to a total volume of 1 ml) was injected intravenously (via the jugular catheter) 15 min before baseline measurements (Kurose et al., 1994) as a single bolus. The recorded fluorescent images were digitized and the gray levels depending on the fluorescence intensity (gray levels ranging from 0 [black] to 255 [white]) were measured within three segments of the venule under study (Iv) and in three continuous areas of the perivenular interstitium (Ii), that were directly attached to the evaluated vessel. Macromolecular leakage was determined as the ratio Ii/Iv at 0, 60, and 120 min. The mean arterial blood pressure was recorded at baseline and after 60 min and 120 min. Statistical analysis At first, the data was evaluated for normal distribution with the Kolmogorov–Smirnov test. All data are presented as mean ± sem. For statistical analysis one way analysis of variance followed by the Bonferroni adjustment procedure were used. Paired samples t-tests were performed to compare variables within one group. Differences were considered significant at p b .05.

Fig. 4. Plasma extravasation of FITC-albumin after burn plasma infusion (BP), burn plasma infusion after additional treatment with cerium nitrate 10 min post trauma (CN10) and 2 h post trauma (CN120) and sham burn plasma infusion (SB). FITC-albumin was progressively observed in the interstitium after burn plasma infusion. Treatment of donor rats with cerium nitrate reduced macromolecular leakage to sham burn levels.

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Results Macrohemodynamic changes No significant differences were observed in mean arterial pressure between the groups at baseline (Sham burn: 107 ± 2 mm Hg; CN10: 121 ± 4 mm Hg; CN120: 122 ± 30 mm Hg; BP: 126 ± 5 mm Hg). During the entire experiment mean arterial blood pressure did not change significantly but stayed basically stable in all groups (120 min: Sham burn: 105 ± 2 mm Hg; CN10: 117 ± 2 mm Hg; CN120: 177 ± 24 mm Hg; BP: 118 ± 4 mm Hg). Differences between groups were not significant. Microhemodynamic changes The effects of plasma infusion on venular diameters, erythrocyte velocity, volumetric blood flow and venular wall shear rate are summarized in Table 1. Venular diameters increased slightly but significantly in the BP and CN120 group over the observation period, whereas diameters did not change significantly in the SB and CN10 group. Differences in venular diameters between groups were not significant. Erythrocyte velocity decreased progressively in all groups. Changes were not significant in the BP and CN10 group, whereas CN120 and SB showed a significant decrease. Differences in erythrocyte velocity between groups were not significant at any time point. Volumetric blood flow did not differ significantly between groups at any time point. It was constant over time in the CN10 and BP groups and decreased slightly but not significantly in CN120 (p N .05), whereas changes in SB were significant (p b .05). During the infusion of the different types of plasma the venular wall shear rate decreased from 579 s− 1 to 369 s− 1 in SB, 577 s− 1 to 414 s− 1 in CN120 and 554 s− 1 to 477 s− 1 in 10% burn plasma. Venular wall shear rate stayed basically stable in the CN10 group (345 s− 1 to 355 s− 1). Differences within groups were not significant in CN10 and BP but were significant in CN120 and SB. Differences between groups were not significant at any time point. Leukocyte sticking The results of leukocyte adherence measurements are demonstrated in Fig. 2. At baseline, there were no significant differences between groups. The number of adherent leukocytes tended to be lower in the SB and BP groups (45.5 ± 32.5 cells/mm2 in SB and 42.4 ± 24.7 cells/mm2 in BP vs. 33.9 ± 23.3 cells/mm2 in CN10 and 26.0 ± 30.1 in CN120; p N .05). New adherent leukocytes, defined as difference between leukocytes that were adherent to the endothelium at baseline and sticking leukocytes at 60 min (15.5 ± 26.7 cells/mm2 in SB, 11.2± 31.9 cells/mm2 in BP, 6.7 ±27.6 cells/mm2 in CN10 and 7.8 ±23.1 in CN120; pN .05) and 120 min (28.6±26.7 cells/mm2 in SB, 30.1 ±31.9 cells/mm2 in BP, 22.8± 27.6 cells/mm2 in CN10 and 17.7±23.1 in CN120; p N .05), increased in all groups. The increase tended to be greater in the SB and BP groups compared to the cerium nitrate treated groups. However, differences were not significant. Differences between SB, CN10, CN120 and BP were not significant, too (Fig. 2). Macromolecular leakage The ratio Ii/Ie did not differ between groups at baseline (SB: 0.11 ± 0.02; CN10: 0.15 ± 0.02; CN120: 0.12 ± 0.03; BP: 0.12 ± 0.01). After infusion of sham burn plasma as well as burn plasma and burn plasma after cerium nitrate treatment macromolecular leakage was significantly increased (Ii/Ie at 120 min SB: 0.36 ± 0.04; CN10: 0.39 ± 0.05; CN120: 0.38 ± 0.11; BP: 0.78 ± 0.05). The increase of the ratio Ii/ Ie was significantly greater in the burn plasma infusion group compared to the other groups. At 60 min, differences between BP and SB were significant. At 120 min, macromolecular leakage in the BP

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Table 2 p-values for differences between groups in terms of albumin extravasation at 120 min (Infusion of sham burn plasma [SB], burn plasma after additional treatment with cerium nitrate 10 min post trauma [CN10] and 2 h post trauma [CN120] and burn plasma without treatment [BP]). SB SB CN10 CN120 BP

p N .05 p N .05 p = .001

CN10

CN120

BP

p N .05

p N .05 p N .05

p = .001 p b .001 p b .001

p N .05 p b .001

p b .001

group was significantly greater compared to all other groups. In contrast, SB, CN10 and CN120 were statistically not different (Figs. 3, 4 and Table 2). Discussion Major burns are still hard to treat and despite modern intensive care medicine burned patients still show considerable mortality rates (Cioffi, 2001; Vogt et al., 2007). Early mortality has classically been addressed to acute circulatory or respiratory failure, whereas late deaths have been ascribed to sepsis (Cioffi, 2001; Garner and Heppell, 2005). However, it has been increasingly recognized, that these deaths may result from immunologic failure in the absence of documented sepsis (Demling, 1985). Early excision of the burn eschar has been shown to improve survival and to reduce late immunologic problems (Herndon and Spies, 2001; Tompkins et al., 1986). This clinical observation reinforces the idea that the burn wound or burn eschar may be responsible for the generation of immunosuppression. Experimental evidence concurs with this view: sterile homogenates of burned mouse skin were injected intraperitoneally in healthy individuals and lead to an 80% mortality whereas unburned skin showed no lethal effect (Allgöwer et al., 1963). However, early excision – and thus removal of the toxic effect of burned tissue – is not always possible due to logistic reasons such as a lack of temporary wound dressings or human resources or due to restrictions of the patient himself (co-morbidities or concomitant injuries) (Ross et al., 1993). Therefore, alternative topical treatment options were developed to find a way to postpone radical excision (Vehmeyer-Heeman et al., 2006). Cerium nitrate, a rare earth element, was introduced in the therapeutic armamentarium in 1976 (Fox et al., 1977; Monafo et al., 1976). In early studies, a reduction of mortality rates was observed compared to a calculated number of deaths that were anticipated (Monafo et al., 1976; Scheidegger et al., 1992). However, prospective randomized trials, which compared topic silver sulfadiazine alone with silver sulfadiazine plus cerium nitrate presented with inconsistent results. de Gracia (2001) showed cerium nitrate to reduce the mortality risk whereas Munster et al. (1980) found both treatment options to be equally effective. The effects of cerium nitrate treatment after thermal trauma on cytokine levels and immunologic activity have been widely investigated (Antonacci et al., 1982; Deveci et al., 2000; Peterson et al., 1985; Sparkes, 1996; Zapata-Sirvent and Hansbrough, 1985). Nevertheless, capillary leakage as a major pathophysiologic endpoint of burn injury has not been evaluated by intravital microscopy after topical cerium nitrate treatment. Intravital microscopy is well established and reliable in the evaluation of local as well as systemic effects of burn injury (Eski et al., 2001; Langer et al., 2005; Reynoso et al., 2007). However, results have to be interpreted carefully. Leukocyte– endothelial interactions as well as macromolecular leakage have been shown to be dependent on differences in microhemodynamic parameters, such as red blood cell velocity and venular wall shear rates (Kubes et al., 1995; Walther et al., 2003, 2000). The mean arterial blood pressure (MAP) was measured in the present study to demonstrate similar macrohemodynamic conditions in all groups. MAP stayed stable during the entire experiment, whereas MAP decreased

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slightly but not significantly in other studies (Kremer et al., 2008; Walther et al., 2000). Microhemodynamic changes showed similar changes as described previously (Kremer et al., 2008). However, differences between groups were not significant, neither in macro— nor in microhemodynamic parameters. These findings demonstrate that the observations in terms of macromolecular leakage and leukocyte adherence can clearly be addressed to effects derived from plasma infusion and are not dependent on significant differences between groups. Our group as well as others were able to demonstrate that burn injury leads to an increased transvascular flux of fluorescence labeled albumin from the intravascular to the extravascular space in remote tissues such as the mesentery (Huang et al., 2003; Kremer et al., 2008; Reynoso et al., 2007). Furthermore, we demonstrated that the transfer of blood plasma from burned rats to healthy individuals leads to systemic burn edema similar to direct thermal injury. This effect was still observed, when burn plasma was diluted (Kremer et al., 2008). In conclusion, together with burn plasma a sufficient amount of plasma factors, such as immunomodulative cytokines, is transferred to induce a cascade that induces systemic burn edema and subsequently burn shock in otherwise healthy individuals. In the present study the infusion of burn plasma in a 10% dilution in saline resulted in a significant increase of macromolecular leakage in the rat mesentery. In comparison, edema formation after sham burn infusion was significantly lower. These results compare with previous results (Huang et al., 2003; Kremer et al., 2008). Interestingly, macromolecular leakage after infusion of plasma from rats after thermal trauma which were additionally bathed in cerium nitrate (0.05 M) was reduced to sham burn plasma infusion levels. Furthermore, this effect was still observed with cerium nitrate treatment of donor rats at 2 h post trauma. These results show that the release of plasma factors from the burn eschar is significantly reduced after cerium nitrate treatment or that cerium nitrate reduces plasma levels in a way that this plasma is not sufficient anymore to induce burn shock in healthy individuals. This positive effect of cerium nitrate treatment on burn edema formation may explain the mortality reduction that was clinically observed (Monafo et al., 1976; Scheidegger et al., 1992). In many burn centers cerium nitrate is used to postpone surgical burn wound excision (Vehmeyer-Heeman et al., 2006) in order to allow sequential débridements in high risk patients to reduce the risk of each procedure (Vehmeyer-Heeman et al., 2007). Patients stay initially more stable after cerium nitrate treatment which – to date – was interpreted as a reduction of wound infection. In the light of the present study these observation may also be explained by a significant reduction of capillary leakage with respective benefit for the patient. However, the underlying mechanisms of the beneficial effects of cerium nitrate treatment have not been explained yet. After thermal injuries a variety of cytokines, prostaglandins and other inflammatory mediators are released (Arturson, 1985, 1996, 2000). Several of these cytokines, such as TNF-alpha and IL-1, have been shown to induce systemic leukocyte and endothelial cell activation following burn injury (Algöwer et al., 1995; Deveci et al., 2000; Endo et al., 1993; Sparkes, 1996; Zhang et al., 1998). This activation results in leukocyte–endothelial cell interactions, which consist of three different sequential steps — leukocyte rolling, sticking, and transmigration (Cioffi et al., 1992; Eski et al., 2001). One potential initiator of this activation was described by Allgöwer et al. who isolated a high molecular weight (3,000,000 Da) “lipid protein complex” formed by heat-induced polymerization of six skin polypeptides (Garner and Heppell, 2005; Schoenenberger et al., 1975). It is suggested that cerium nitrate may fix LPC in the burn eschar tissue and may therefore reduce leukocyte activation after burn injury. Eski et al. described a significant increase of rolling, adherent and transmigrating leukocytes after burn injury that was not observed after sham burn. Cerium nitrate bathing immediately after burn injury for

30 min reduced leukocyte activation (rolling, sticking and transmigration) to baseline levels (Eski et al., 2001). In contrary, in our model leukocyte adherence was not significantly influenced by cerium nitrate treatment. Nevertheless, leukocyte sticking showed a trend to lower activation after cerium nitrate treatment. The differences between studies may be explained by different models (rat mesentery vs. cremaster muscle) or by different ways to calculate adherent leukocytes (cells/mm2 vs. cells in a part of a postcapillary venule). The fact that leukocyte adherence is not significantly influenced by cerium nitrate treatment in the present model and – in contrary – macromolecular leakage is reduced significantly after cerium nitrate bathing shows, that burn edema formation seems to be independent from leukocyte activation in the present model. This mechanism has already been suggested in previous studies and has been described in sepsis and endotoxemia already (Walther et al., 2000). But how does cerium nitrate exert this potent effect in terms of burn edema reduction? One potential effect is that cerium nitrate renders the eschar firm and impermeable, often described as leatherlike appearance (Boeckx et al., 1992; Garner and Heppell, 2005). Furthermore, Boeckx et al. postulated in an histologic study, that cerium may bind tissue pyrophosphate and therefore reduce the negative immunomodulatory effect of the burn eschar (Boeckx et al., 1992). Other studies showed, that cerium nitrate directly binds LPC and may reduce burn related immunosuppression and burn edema this way. In conclusion, the beneficial mechanism of cerium nitrate treatment is not entirely clear to date, however several studies have proven cerium nitrate treatment to reduce the pathologic consequences of thermal injury. In conclusion, burn plasma transfer from rats after burn injury induces burn edema in healthy individuals. Therefore, high concentrations of plasma factors are released in burn victims that are sufficient to induce capillary leakage. Cerium nitrate bathing reduces this plasma factors significantly in a way that burn edema after burn plasma transfer can be prevented. This effect explains the reduction of mortality rates that was observed in clinical studies. The underlying mechanisms of this beneficial effect are not entirely clear but may be physical hardening, which generally makes the eschar impermeable for any pathogen or may be direct binding of cerium nitrate to burn toxins, such as LPC. Cerium nitrate bathing did not significantly influence leukocyte activation in the present model, which shows that burn edema after burn plasma transfer is likely to be independent from leukocyte activation. References Algöwer, M., et al., 1995. Burning the largest immune organ. Burns 21, S7–S47. Allgöwer, M., et al., 1963. Toxicity of burned mouse skin in relation to burn temperature. Surg. Forum. 14, 37–39. Antonacci, A.C., et al., 1982. T-cell subpopulations following thermal injury. Surg. Gynecol. Obstet. 155, 1–8. Arturson, G., 1985. Neutrophil granulocyte functions in severely burned patients. Burns 11, 309–319. Arturson, G., 1996. Pathophysiology of the burn wound and pharmacological treatment. The Rudi Hermans lecture 1995. Burns 24, 309–319. Arturson, G., 2000. Forty years in burns research — the postburn inflammatory response. Burns 26, 599–604. Boeckx, W., et al., 1992. Effect of cerium nitrate–silver sulphadiazine on deep dermal burns: a histological hypothesis. Burns 18, 456–462. Carlson, D.L., et al., 2002. Burn plasma mediates cardiac myocyte apoptosis via endotoxin. Am. J. Physiol. Heart. Circ. Physiol. 282, H1907–H1914. Cioffi, W.G., 2001. What's new in burns and metabolism? J. Am. Coll. Surg. 192, 241–254. Cioffi, W.C., et al., 1992. Leukocyte responses to injury. Arch. Surg. 112, 860–865. de Gracia, C.G., 2001. An open study comparing topical silver sulfadiazine and topical silver sulfadiazine–cerium nitrate in the treatment of moderate and severe burns. Burns 27, 67–74. Demling, R.H., 1985. Burns. N. Engl. J. Med. 313, 1389–1398. Deveci, M., et al., 2000. Effects of cerium nitrate bathing and prompt burn wound excision on IL-6 and TNF-alpha levels in burned rats. Burns 26, 41–45. Endo, S., et al., 1993. Plasma tumor necrosis Factor-alpha levels in patients with burns. Burns 19, 124–127. Eski, M., et al., 2001. Treatment with cerium nitrate bathing modulate systemic

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