Annals of Burns and Fire Disasiers - vol. X1 - n. 4 - December 1998

CERIUM NITRATE BATHING PREVENTS TNF-a ELEVATION FOLLOWING BURN INJURY (EXPERIMENTAL STUDY)

Sengezer M, Deved M, Eski M.

Gulhane Military Medical Academy and Medical Faculty, Department of Plastic and Reconstructive Surgery, Ankara, Turkey

SUMMARY. The physiopathological changes in the burn wound are not only characterized by the effects of heat but are also closely related to pronounced acute inflammatory processes. Following thermal injury, induction of a number of cytokines rapidly occurs. TN17-ot may be considered to be the most important cytokine related to systemic inflammation and multiple organ failure after a major trauma. Using cerium nitrate, the improvement in survival has been reported to be equal to that observed following prompt excision of the burn eschar. The aim of this experimental study was to investigate the effectiveness of cerium nitrate bathing with respect to serum levels of TNF-a in burned rats. It was observed that treatment with cerium nitrate prevented the elevation of TNF-a levels in the early stages of thermal injury. This would appear to prevent toxic materials originating in the burn wound from entering the blood stream and thus to improve survival.

Introduction

Physiopathological changes in burn wound are not only characterized by the effects of heat but are also closely related to pronounced acute inflammatory processes.' Following thermal injury, the induction of a number of cytokines rapidly occurs. The overproduction of some cytokines and the activation of leukocytes and endothelial cells may cause an abortive production of active inflammatory substances. This series of events may lead to the systemic inflammatory response syndrome (SIRS), the acute respiratory distress syndrome (ARDS), or the multiorgan dysfunction syndrome (MODS), which may develop further into progressive organ failure and death." According to Arturson, the burn eschar should be regarded as a specialized "organ", one that can communicate with the body and influence a variety of systemic circulatory and metabolic changes. Communication between host and burned tissue is known to be managed by cytokines.Cytokines are intercellular signal proteins or peptides that modulate the inflammatory response following trauma. They are considered to be important as regulators of resistance to infections in patients with thermal injury. The most important cytokines are IL-2, IL-6 and TNF-a. in burn patients. TNF-a is mainly produced by activated macrophages. TNF-a regulates the production of some other cytokines. It also enhances endothelial adhesiveness for leukocytes and stimulates neutrophils and monocytes, promoting their adherence, phagocytosis, oxidative burst, and degranulation. Since it has a powerful effect on inflammation, TNF-a may be considered to be the most important cytokine related to systemic inflammation and multiple organ failure following major trauma. Both TNF-a and IL-6 are thought to be important indicators of poor prognosis after thermal injury.
Since the mid-1970s cerium nitrate, a rare earth element, has been reported to be effective on burn wounds. It has antiviral activity in vitro and is relatively nontoxic. Monafo et al. reported that cerium nitrate also improves survival in burn patients when used topically. Using cerium nitrate, the improvement in survival has been reported to be equal to that observed after prompt excision of the burn eschar. Cerium nitrate has been reported to bind trivalently the lipid-protein complexes that are originated from burned tissue. Topical application or bathing with cerium nitrate thus prevents the toxic material from contaminating the blood stream and neutralizing the inurnmosuppressive effect. Scheidegger et al. reported that treatment within one day post-burn, with one single bathing of not less than thirty minutes, is a simple way of neutralizing toxic eschar material and of improving survival! The aim of this experimental study was to investigate the effectiveness of cerium nitrate bathing with respect to TNF-a serum levels in burned rats.

Materials and methods

Forty male Sprague-Dawley rats weighing between 200 and 250 g were obtained from the Gulhane Military Medical Academy Experimental Research Centre and housed in individual cages at 28 ± 2 °C and approximately 40% relative humidity, with a 12/12 h light-dark cycle. The rats were given standard rat chow and water ad libitum.

Procedure
The rats were anaesthetized by inhalation of sevofluorane, and the hair was removed from the dorsal surface by shaving with animal epilatory agent. The dorsal area was scrubbed with Betadine twice following hair removal. The experimental area was then cleansed with 70% ethanol. Burn injuries were inflicted by submerging the dorsal area of the animals in water at 90 °C for 20 see. This resulted in full-thickness burn wounds (mean size, 20 ± 5 % TBSA). All animals received lactated Ringer's solution i.p. equal to 18% body weight and a comparable period of anaesthesia. Forty male Sprague-Dawley rats were randomly separated as experimental and control groups. The experimental group of rats received single bathing in cerium nitrate for 30 min immediately after thermal injury. Burn wounds in the dorsal area were left undressed. Rats in the control group received no treatment following the burn injury. Each group was divided into two subgroups on the third and seventh days following the burn.Blood samples were collected on days 3 and 7 postburn. The scrum specimens collected after centrifugation were stored at -70 °C until they were analysed. The data were collected and processed using software provided by SPSS (Statistical Package for Social Sciences, Microsoft). The statistical analysis was performed using Student's t test.

TNF-a assay
The enzyme-linked immunosorbent assay (ELISA) was performed using the CytoScreen Rat TNF-a. Kit (BioSource, Camarillo, Calif.). The Cytoscreen Rat TNF-a Kit is a solidphase sandwich ELISA. An antibody specific for TNF-a was coated onto the wells of microtitre strips provided. Samples (including standards of known TNF-a content), control specimens and unknowns were pipetted into these wells. This was followed by the addition of biotinylated second antibody. During the first incubation, the TNF-a antigen binds simultaneously to the immobilized antibody on one site and to the solution phase biotinylated antibody on a second site. After removal of excess second antibody, the enzyme streptavidin-peroxidase is added. This binds to the biotinylated antibody to complete the four-member sandwich. After a second incubation and washing to remove all unbonded enzymes, a substrate solution is added, which is acted upon by the bound enzyme to produce colour. The intensity of this coloured product is directly proportional to the concentration of TNF-a present in the original specimen. The plates are read spectrophotometrically in a microplate reader at 450 nm.

Results

On the third day post-burn, TNF-a levels in the control group were found to be statistically different when compared with those in the experimental group. The mean TNF-a level in the control group was found to be 24.6. The mean TNF-a level in the experimental group was 19.3. The difference was statistically significant (p < 0.001). On the seventh day post-burn, TNF-a levels in the control group were again found to be statistically different when compared with those of the experimental group. The mean TNF-a level in the control group was 25.4. The mean TNF-a level in the experimental group was 14.6. The difference was again statistically significant (p < 0.001).

Discussion

The burn is the most devastating injury to the body. Mortality from severe thermal injury is reported to have occurred in over 50% of patients without any evidence of sepsis. Clinical and experimental studies have shown that post-burn immunosuppression makes the patients more susceptible to infection. Sepsis is not however the most important cause of death following thermal injury. Sparkes reported that the skin is an immune organ that causes post-burn pathophysiological events following thermal injury. The skin has been reported as being a triggering part of the body when exposed to thermal injury, especially when early excision is not performed. In an experimental study, it was found possible to kill mice applying a patch of burned skin to the skinless area of mice from which it had been removed. This indicates that the absorption of toxic material from the burned skin into the circulation was an event that triggered pathophysiological consequences. It is generally accepted that early tangential excision and grafting procedures strongly increase the survival prospects of burn patients. This cannot simply be explained by the prevention of contamination, although this procedure is the most effective way to prevent the absorption of toxic materials that affect the host-immune response.
The pathophysiological changes in the burn wound are characterized by effects caused by heat per se, upon which a pronounced acute inflammation process is superimposed. Increases in body temperature cause a severe inflammatory reaction mediated by the local release of inflammatory mediators and cascades of reactions. Cytokines are intercellular signal proteins or peptides that modulate the inflammatory response following thermal injury and they are considered to be important as regulators of the resistance to infection in burn patients. An inflammatory reaction to injury, an antigen challenge with overproduction, the activation of leukocytes and endothelial cells, alteration in circulating cytokines - all these may contribute to the systemic effect. In patients with severe thermal injury, this inflammation may therefore cause SIRS, and eventually MODS and death. Death subsequent to thermal injury must thus be considered to be related to improper systemic and inflammatory reactions, as a result of which the patient eventually ends up destroying himself, using his own inflammatory system. Cytokines are the most important mediators of this inflammatory reaction. Among these, IL-2, IL-6 and TNF-a are the most effective. Sparkes has defined the events in the cytokine cascade as the "chaos" followig thermal injury. 2 Boeckx et al. reported that the topical application of cerium nitrate and silver sulphadiazine cream causes superficial calcification on the burn wound and thus acts as a preventive shell.' This phenomena is termed "calciphylaxis" by several authors. The topical application of cerium nitrate is reported to bind inorganic pyrophosphate and precipitate together, causing the sealing-off of deep dermal burns. It is reported that treatment with cerium nitrate causes a basophilic band in the deep dermal layer and a relative lack of leukoeytic infiltration. It has therefore been postulated that the occurrence of a shell might constitute a physical barrier that insulates the exposed collagen from the environmentafeffects of micro-organisms and other physical conditions. Changes in the cytokine cascade and cellular immune system following thermal injury have been widely investigated. However, these effects have yet to be fully interpreted. We observed that treatment with cerium nitrate bathing prevents the elevation of TNF-a levels in the early stages of thermal injury. Advances in technology and recent experimental studies have considerably modified our understanding of post-burn immune failure following thermal injury. The most important event in burn injury is the interaction between host and burn eschar. As Arturson stated, the burn eschar must be regarded as a specialized organ, an organ that communicates with the host. This communication can be interrupted by removal of the burn eschar or by the constitution of a barrier by means of agents such as cerium nitrate. Avoidance of this type of interaction between host and burn eschar results in the prevention of triggering systems, Severe inflammatory response syndrome can be prevented in this way. The literature in recent years has presented numerous papers describing burn patients successfully treated with cerium nitrate, either by bathing or by topical application. In conclusion, it was observed that a single session of cerium nitrate bathing can prevent the elevation of TNF-a in burned rats. This would appear to prevent toxic materials originating in the burn wound from entering the blood stream and thus improve survival.

RESUME. Les modifications physiopathologiques qui se produisent à cause des brûlures ne sont pas caracterisées seulement par les effets de la chaleur mais elles sont aussi corrélées avec un processus inflammatoire aigu marqué. A la suite d'une brûlure il se produit rapidement l'induction de diverses cytokines. On peut considérer la TNF-a. la cytokine la plus importante pour ce qui concerne l'inflammation systémique et l'insuffisance multiorganique après un grave traumatisme. Avec l'emploi du nitrate de cérium, l'amélioration de la survivance s'est démontrée égale après l'excision de l'escarre. Dans cette étude expérimentale les Auteurs se sont proposés de considérer l'efficacité du traitement moyennant le nitrate de cérium par rapport aux niveaux sériques de TNF-a. dans les rats brûlés. Les Auteurs ont observé que le traitement avec des lavages de nitrate de cérium réduit l'élévation des niveaux de TNF-a dans les premières phases des lésions thermiques. Cet effet semble bloquer les matériaux toxiques provenant de la brûlure, qui n'entrent pas dans la circulation sanguine, avec des résultats positifs pour les possibilités de survivance.

BIBLIOGRAPHY

1. Arturson G.: Pathophysiology of the burn wound and pharmacological treatment. The Rudi Herman Lecture, 1995. Burns, 22: 255, 1996. 
2. Sparkes B.G.: Immunological responses to thermal injury. Burns, 23: 106, 1997.
3. Munster A.M.: The immunological response and strategies for intervention. In: Hemdon D. (ed.), "Total Burn Care", pp. 279-92. W.B. Saunders Co., Philadelphia, 1996.
4. Sparkes B.G_ Gyorkos JW., Gorczynsky R.M., Brock A.J.: Comparison of endotoxins and cutaneous burn toxin as immunosuppressants. Burns, 16: 123, 1990.
5. Alexander JW.: Mechanism of immunologic suppression in burn injury. J. Trauma, 30: 70, 1990
6. SchindlerR., MancilliaJ., Endres S., Ghorbani R., Clark S.C., Dinarello C.A.: Correlations and interactions in the production of interleukin-6 (IL-6), IL- I and tumor necrosis factor (TNF) in human blood mononuclear cells: IL-6 suppresses IL- I and TNF. Blood, 75: 40, 1990.
7. Scheidegger D., Sparkes B.G., Ldscher N., Schoenenberger G.A., AlIg6wer M.: Survival in major burn injuries treated by one bathing in cerium nitrate. Burns, 18: 296, 1992.
8. Boeckx W., Blondeel N., Vandersteen K., De Wolf-Peeters C., Schmitz A.: Effect of cerium nitrate-silver sulphadiazine on deep dermal burns: a histological hypothesis. Burns, 18: 456, 1992.
9. Monafo W.W., Tandon S.N., Ayvazian VA.: Cerium nitrate: a new topical antiseptic for extensive burns. Surgery, 80: 465, 1976.
10. Peterson V.M., Hansbrough J.F., Wang XW., Zapata-Sirvent R., Boswick J.A.: Topical cerium nitrate prevents postburn immunosuppression. J. Trauma, 25: 1039, 1985.
11. Sparkes B.G., Monge G., Marshall S.L ., Peters W.J., Allg6wer M., Schoenenberger G.A.: Plasma levels of cutaneous burn toxin and lipid peroxides in thermal injury. Burns, 16: 118, 1990.
12. Caldwell F.T., Graves D.B., Wallace B.H.: Pathogenesis of fever in a rat burn model: the role of cytokines and lipopolysaccharide. J. Burn Care Rehabil., 18: 525, 1997.
13. Zhou D., Munster A.M., Winchurch R.A.: Inhibitory effects of interleukin-6 on immunity. Possible implications in burn patients. Arch. Surg., 127: 65, 1992.