The importance of BMPs and TGF-ßs for endochondral bone repair - A longitudinal study in hip arthroplasty patients.

Introduction: Osseointegration of hip implants, although a decade-long process, shows striking similarities with the four major phases of endochondral bone repair. In the current study we investigated the spatiotemporal involvement of bone morphogenic proteins (BMPs) and transforming growth factor betas (TGF-ßs) throughout the process of bone repair leading to successfully osseointegrated hip implants. Materials and methods: Twenty-four patients that had undergone primary total hip arthroplasty (THA) due to one-sided osteoarthritis (OA) were investigated during a period of 18 years (Y) with repeated measurements of plasma biomarkers as well as clinical and radiological variables. All implants were clinically and radiographically well anchored throughout the follow-up. Eighty-one healthy donors divided in three gender- and age-matched groups and twenty OA patients awaiting THA, served as controls. Plasma was analyzed for BMP-1, -2, -3, -4, -6, -7 -9 and TGF-ß1, -ß2, -ß3 by use of a high-sensitivity and wide dynamic range electrochemiluminescence technique allowing for detection of minor changes. Results: Spatiotemporal changes during the follow-up are presented in the context of the four phases of endochondral bone repair shown in earlier studies and transposed to the current study based on similarities in biomarker responses. Phase 1: Primary proinflammatory phase lasting from surgery until day 7, Phase 2: Chondrogenic phase from day 7 until 18 months postsurgery, Phase 3: Secondary proinflammatory and cartilage remodeling phase lasting from 18 months until 7Y, Phase 4: coupled bone remodeling from 7Y until 18Y postsurgery. BMP-1 increased sharply shortly after surgery and remained significantly above healthy during the chondrocyte recruitment, proliferation, and hypertrophy phases with a subsequent return to control level at 5Y postsurgery. BMP-2 was above healthy controls before surgery and 1 day after surgery before decreasing to control level and remaining there throughout the follow-up. BMP-3 was at control level from presurgery until 6M after surgery when it increased to a peak at 2Y during the cartilage hypertrophy phase followed by a gradual decrease to control level at 10Y during the phase of bone formation. In the following, BMP-3 decreased below controls to a nadir 15Y postsurgery during coupled bone remodeling. BMP-4 was at control level from presurgery until 10Y postsurgery when it increased to a sharp peak at 15Y after surgery followed by a return to the level of healthy at 18Y. BMP-6 did not differ from healthy during the follow-up. BMP-7 was at control level from presurgery until 1Y postsurgery before gradually increasing to a peak at 10Y during the early phase of osteogenesis with a gradual return to control level at 18Y during the phase of coupled bone remodeling. BMP-9 was above OA before surgery followed by a decrease to basal level on day 1 after surgery and a renewed increase to a plateau above controls lasting from 6 W until returning to the level of healthy at 18Y postsurgery, i.e., throughout the phases of cartilage formation, cartilage hypertrophy and remodeling, bone formation and coupled bone remodeling. TGF-ß1 was above controls presurgery before decreasing to baseline shortly after surgery followed by a renewed increase at 6 M to a peak at 2Y during cartilage hypertrophy/remodeling followed by a gradual return to baseline at 10Y during early osteoblastogenesis. TGF-ß2 was at control level from presurgery until the phase of cartilage remodeling at 5Y when it increased sharply to a peak at 7Y with a gradual return to baseline at 18Y postsurgery. TGF-ß3 remained at control level throughout the study. Conclusion: This study shows that the involvement of BMPs and TGF-ßs in endochondral bone repair is a process of stepwise recruitment of individual biomarkers characterized by distinct, yet overlaping, spatiotemporal patterns that extend from the early phase of pre-chondrocyte recruitment until the late phase of coupled bone remodeling.

Concerted actions by MMPs, ADAMTS and serine proteases during remodeling of the cartilage callus into bone during osseointegration of hip implants.

INTRODUCTION: Although the number of patients undergoing total hip arthroplasty is constantly on the rise, we only have limited knowledge of the molecular mechanisms necessary for successful osseointegration of implants or the reasons why some fail. Understanding the spatiotemporal characteristics of signaling pathways involved in bone healing of implants is therefore of particular importance for our ability to identify factors causing implants to fail. The current study investigated the role of three families of proteases, i.e. MMPs (matrix metalloproteinases), ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) and serine proteases, as well as their endogenous inhibitors during osseointegration of hip implants that have endured two decades of use without clinical or radiological signs of loosening. MATERIALS AND METHODS: Twenty-four patients that had undergone primary THA due to one-sided osteoarthritis (OA) were monitored during 18 years (Y) with repeated measurements of plasma biomarkers, clinical variables and radiographs. All implants were clinically and radiographically well-fixed throughout the follow-up. Eighty-one healthy donors divided in three gender and age-matched groups and twenty OA patients awaiting THA, served as controls. Plasma was analyzed for MMP-1, -2, -3, -8, -9, -10, -13, -14, tissue inhibitor of metalloproteinase (TIMP)-1, -2, -3, ADAMTS4, ADAMTS5, the serine proteases neutrophil elastase (NE), proteinase 3 (PR3) and their endogenous inhibitors, secretory leucocyte proteinase inhibitor (SLPI), trappin-2/elafin and serpina1 (α-1 antitrypsin). Cartilage turnover was monitored using two markers of cartilage synthesis, type II procollagen and PIICP (procollagen II C-terminal propeptide), and two markers of cartilage degradation, CTX-II (C-terminal telopeptide fragments of type II collagen) and split products of aggrecan (G1-IGD-G2). RESULTS: MMP-1, MMP-9, ADAMTS4, NE and PR3 were above healthy in presurgery OA patients but returned to the level of healthy within 6 weeks (W) after surgery. MMPs and serine proteases were counter-regulated during this phase by TIMP-1, SLPI and trappin-2/elafin. Type II procollagen, PIICP and CTX-II increased to a peak 6 W after surgery with a gradual return to the level of controls within weeks. Significant increases by MMP-8, MMP-9, ADAMTS4, ADAMTS5, NE, PR3 and the protease inhibitors, TIMP-3 and serpina1, were seen 5 Y after hip arthroplasty paralleled by a sharp increase in the levels of the cartilage degradation markers, CTX-II and G1-IGD-G2. All the above mediators were normalized before 18 Y, except MMP-1 and MMP-9 that remained above healthy at 18 Y. MMP-14 increased immediately after surgery and remained elevated until 5 Y postsurgery before returning to the level of controls at 7 Y. CONCLUSION: Notwithstanding temporal differences, the molecular processes of bone repair in arthroplasty patients show great spatial similarities with the classical phases of fracture repair as previously shown in animal models. Cartilagenous callus, produced and remodeled early after hip arthroplasty, is replaced with bone 5 Y to7 Y after surgery by the concerted actions of MMP-8, MMP-9, ADAMTS4, ADAMTS5, NE and PR3, thus suggesting that a complex regulatory cross-talk may exist between different families of proteases during this transitional phase of cartilage degradation. Regulation and fine-tuning of cartilage remodeling by MMPs and ADAMTS is controlled by TIMP-3 whereas serine proteases are regulated by serpina1. Increased MMP-1 and MMP-9 beyond 10Y post-THA support a role during coupled bone remodeling.

The key role of proinflammatory cytokines, matrix proteins, RANKL/OPG and Wnt/ß-catenin in bone healing of hip arthroplasty patients.

INTRODUCTION: We still lack understanding of why some implants fail while most remain stable after decades of use. Proinflammatory cytokines, matrix proteins and bone regulating cytokines of the RANKL/OPG (receptor activator of nuclear factor kappa B ligand/osteoprotegerin) and Wnt/ß-catenin pathways are mandatory for normal bone repair but their spatial and temporal role in the healing of primary total hip arthroplasties (THA) has not been previously shown. MATERIALS AND METHODS: Twenty-four osteoarthritis patients with one-sided well-fixed primary THA were prospectively monitored during 18years (18Y) with repeated blood samples, clinical variables and radiographs. Eighty-one healthy donors divided in three age- and gender-matched groups and twenty osteoarthritis patients awaiting THA and serving as control of the validity of stored plasma in THA patients, were included. Plasma was analyzed for C-reactive protein (CRP), interleukin (IL)-6, IL-8, IL-1ß, tumor necrosis factor (TNF)-α, osteopontin (OPN), secreted protein acidic and rich in cysteine (SPARC/osteonectin), osteocalcin (OC), bone specific alkaline phosphatase (BALP), N-terminal propeptide of collagen type I (P1NP), RANKL, OPG, the Wnt agonistic ligands (Wnt)-1 and Wnt-3a, and the Wnt antagonists sclerostin, Dickkopf (Dkk)-1, Dkk-3, Dkk-4, secreted frizzled related protein (sFRP)-1, sFRP-3 and Wnt inhibitory factor-1 (Wif-1). RESULTS: Inflammatory mediators in arthroplasty patients (CRP, IL-6, OPN) increased significantly on day one after surgery vs preoperative value (PR) and healthy subjects and returned to baseline at 6W. TNF-α did not change relative preoperative level or healthy subjects. SPARC and OC increased in a biphasic fashion with the primary phase beginning shortly after surgery and lasting 3M (SPARC) and 2Y (OC) while the secondary phase peaked at 1Y (SPARC) and 13Y (OC), with both returning to basal level at 15Y. BALP peaked at 3M after surgery with a return to basal level at 2Y followed by a continuous increase from 5Y until 18Y. P1NP increased immediately after surgery and returned to basal level at 6W followed by a new peak at 10Y returning to basal at 13Y. IL-8 and IL-1ß peaked at 5Y post-THA and returned to basal level at 10Y. RANKL/OPG and Wnt/ß-catenin remained at preoperative levels until 5Y post-THA when a sustained increase in OPG level, paralleled by a sustained decrease in sclerostin, started and lasted until 18Y. Despite a strong increase by RANKL at 13Y, the OPG/RANKL-ratio remained high between 5Y and 18Y. Dkk-1 and sFRP-1 remained at basal level until 5Y followed by a peak at 7Y and a return to basal level at 15Y. Similarly, RANKL increased after 5Y, peaked at 13Y and returned to basal levels at 18Y, thus coinciding with Wnt-1. In contrast, Wnt3a, Dkk-3, Dkk-4, sFRP-3 and Wif-1 did not differ from preoperative levels or healthy subjects during the course of the follow-up. CONCLUSION: The primary peak of proinflammatory cytokines involved in the initiation of bone healing after trauma is in line with previous results. The primary phase of increased matrix proteins, P1NP and BALP paralleled by RANKL, OPG and Wnt/ß-catenin remaining at preoperative level until 5Y, support a strong formation of mineralized matrix and to a lesser degree bone during this phase. The secondary proinflammatory peak at 5Y is likely a trigger of coupled bone remodeling and neosynthesis as it is followed by increased levels of the bone anabolic turnover marker, BALP, and mediators of the RANKL/OPG and Wnt/ß-catenin pathways. A continuous increase by OPG level and the bone turnover marker, BALP, lasting from 5Y until 18Y and paralleled by a similar decrease in sclerostin level support their being key regulators of bone anabolism, whereas the transient and opposed activities of RANKL, Wnt-1, Dkk-1 and sFRP-1 serve as fine tuning tools during the coupled remodeling phase.

Offloading treatment is linked to activation of proinflammatory cytokines and start of bone repair and remodeling in Charcot arthropathy patients.

BACKGROUND: Proinflammatory cytokines are an integral part of the osteolytic activity of Charcot arthropathy but are also central to normal bone healing. As there are no previous longitudinal studies investigating their role during the recovery phase of Charcot, we set out to monitor systemic levels of proinflammatory cytokines from Charcot presentation until a clinically and radiographically documented chronic state has been reached. METHODS: Twenty-eight consecutive Charcot patients were monitored during 2 years by repeated foot radiographs, MRI and plasma levels of interleukin [IL]-6, IL-8, IL-1ß, Tumor Necrosis Factor [TNF]-α, and IL-1 receptor antibody (IL-1RA). Charcot patients were treated with total contact cast (TCC) on the first day of inclusion. Neuropathic diabetic controls (n = 20) and Healthy subjects (n = 20) served as reference. RESULTS: Plasma IL-6, IL-8, IL-1ß and TNF-α in the acute and chronic phase of Charcot were below or at the level of diabetic controls and healthy, whereas IL-1RA/IL-1ß ratio was continuously higher in Charcot patients. IL-6, TNF-α and IL-1RA began to increase one week after offloading to reach a peak after 4 months before gradually receding. CONCLUSIONS: A sustained increase of IL-6 and TNF-α starting shortly after offloading and paralleled by accelerated bone healing on radiographs, suggest that offloading, by activating the inflammatory stage, has a key role to play in the onset of coupled bone remodeling. High IL-1RA/IL-1ß ratio in Charcot patients at presentation supports a counter-balancing anti-inflammatory role for IL-1RA in the acute phase whereas a high ratio after two years, possibly due to renewed weight-bearing on a deformed foot, signal need for continued anti-inflammatory activity and contradicts a "cold" biological state in the chronic phase.

IL-17 cytokines in bone healing of diabetic Charcot arthropathy patients: a prospective 2 year follow-up study.

BACKGROUND: Little is currently known of the pathophysiological mechanisms triggering Charcot arthropathy and regulating its recovery although foot trauma has been proposed as a major initiating factor by activation of proinflammatory cytokines leading to increased osteoclastogenic activity and progressive bone destruction. Several members of the IL-17 family of proinflammatory cytokines have been shown to play a key role in the pathogenesis of inflammatory conditions affecting bone and joints but none has previously been studied in Charcot foot patients. The aim of this study was to investigate the role of IL-17A, IL-17E and IL-17F in patients presenting with Charcot foot. METHODS: Twenty-six consecutive Charcot patients were monitored during 2 years by repeated foot radiographs, MRI and circulating levels of IL-17A, IL-17E and IL-17F. Analysis of cytokines was done by ultra-sensitive chemiluminescence technique and data were analyzed by one-way repeated measures ANOVA. Neuropathic diabetic patients (n = 20) and healthy subjects (n = 20) served as controls. RESULTS: Plasma IL-17A and IL-17E in weight-bearing Charcot patients at diagnosis were at the level of diabetic controls, whereas IL-17F was significantly lower than diabetic controls. A significant increase in IL-17A and IL-17E reaching a peak 2-4 months after inclusion and start of offloading treatment in Charcot patients was followed by a gradual decrease to the level of diabetic controls at 2 years postinclusion. In contrast, IL-17F increased gradually from inclusion to a level not significantly different from diabetic controls after 2 years. CONCLUSIONS: Charcot patients display a significant elevation of all three IL-17 cytokines during the follow-up period relative values at diagnosis and values in control patients supporting a role in the bone repair and remodeling activity during the recovery phase. The rapid increase of IL-17A and IL-17E shortly after initiating off-loading treatment could suggest this to be a response to immobilization and stabilization of the diseased foot.

Role of Wnt/ß-catenin and RANKL/OPG in bone healing of diabetic Charcot arthropathy patients.

BACKGROUND AND PURPOSE: Charcot neuropathy is characterized by bone destruction in a foot leading to deformity, instability, and risk of amputation. Little is known about the pathogenic mechanisms. We hypothesized that the bone-regulating Wnt/ß-catenin and RANKL/OPG pathways have a role in Charcot arthropathy. PATIENTS AND METHODS: 24 consecutive Charcot patients were treated by off-loading, and monitored for 2 years by repeated foot radiography, MRI, and circulating levels of sclerostin, dickkopf-1, Wnt inhibitory factor-1, Wnt ligand-1, OPG, and RANKL. 20 neuropathic diabetic controls and 20 healthy controls served as the reference. RESULTS: Levels of sclerostin, Dkk-1 and Wnt-1, but not of Wif-1, were significantly lower in Charcot patients than in the diabetic controls at inclusion. Dkk-1 and Wnt-1 levels responded to off-loading by increasing. Sclerostin levels were significantly higher in the diabetic controls than in the other groups whereas Wif-1 levels were significantly higher in the healthy controls than in the other groups. OPG and RANKL levels were significantly higher in the Charcot patients than in the other groups at inclusion, but decreased to the levels in healthy controls at 2 years. OPG/RANKL ratio was balanced in all groups at inclusion, and it remained balanced in Charcot patients on repeated measurement throughout the study. INTERPRETATION: High plasma RANKL and OPG levels at diagnosis of Charcot suggest that there is high bone remodeling activity before gradually normalizing after off-loading treatment. The consistently balanced OPG/RANKL ratio in Charcot patients suggests that there is low-key net bone building activity by this pathway following diagnosis and treatment. Inter-group differences at diagnosis and changes in Wnt signaling following off-loading treatment were sufficiently large to be reflected by systemic levels, indicating that this pathway has a role in bone remodeling and bone repair activity in Charcot patients. This is of particular clinical relevance considering the recent emergence of promising drugs that target this system.

[Disaster planning--lessons learned from the fire disaster in Gothenburg]. / Beredskap för det oväntade--lärdomar från brandkatastrofen i Göteborg.

The tragic fire disaster in Sweden 1998, killing 63 and wounding 213 teenagers was the result of arson fire in the basement of an overcrowded discotheque. Since the disaster occurred in a major city with substantial resources, all the injured could be hospitalised within two hours. The load on four local hospitals was initially severe due to the large number of injured. The patients suffered from inhalation injuries of different severity, whereas 25 of them also had deep skin burn injuries. Thirteen of the most severe burn injuries were transported to burn units in other parts of Sweden and to Norway. The vast psychosocial rehabilitation program initiated by health care staff, religious communions, schools and the community, has continued over the past years. This fire disaster emphasises the need for extensive preparation not only in the rescue and medical services, but also in many other areas of society.

Adrenoceptor subtypes in the control of burn-induced plasma extravasation.

Burn trauma is known to induce a significant rise in circulating catecholamine levels and despite catecholamines being potent endogenous vasoactive agents with known actions on microvascular permeability, their effect on burn edema has been poorly investigated. The present study in rats investigated the role and importance of adrenergic receptor subtypes in the regulation of basal capillary permeability in normal skin and hyperpermeability in partial- and full-thickness skin burns. Edema was quantified by spectrophotometric analysis of extravasated Evans blue-albumin. Evaluation was based on intravenous administration of the following adrenergic agonists and antagonists: l-phenylephrine (alpha(1)-receptor agonist), prazosin (alpha(1)-receptor antagonist), clonidine (alpha(2)-receptor agonist), yohimbine (alpha(2)-receptor antagonist), prenalterol (beta(1)-receptor agonist), terbutaline (beta(2)-receptor agonist), or propranolol (beta(1)- and beta(2)-receptor antagonist). Results showed increased capillary permeability in normal skin following administration of terbutaline (p<0.01) and yohimbine (p<0.01). In partial-thickness burns, clonidine significantly (p<0.05) reduced edema formation, whereas in full-thickness burns edema was significantly reduced by clonidine (p<0.05) and l-phenylephrine (p<0.01). In conclusion, the inhibition of postburn edema induced by stimulation of alpha(1)-receptors (l-phylephrine) and alpha(2)-receptors (clonidine) could be secondary to increased vascular resistance and reduced tissue perfusion pressure and/or suppressed inflammatory reaction in the burn injury. In the treatment of burn patients, clonidine is particularly interesting since the agent has previously been proven to induce potent analgesia in thermally injured.

Regulation of postburn ischemia by alpha- and beta-adrenoceptor subtypes.

Deep skin burns are characterised by progressive ischemia secondary to vasoconstriction and thrombosis formation. Burn trauma elicits increased sympathetic activity and elevation of circulating catecholamines acting on adrenoceptors in vascular tissue playing an important role in the regulation of organ blood flow. The present study in rats investigated the role of alpha- and beta-adrenoceptors in the circulatory changes taking place in normal skin and in partial- and full-thickness skin burns using laser Doppler flowmetry. Evaluation was based on intravenous administration of the following adrenergic agonists and antagonists: l-phenylephrine (alpha(1)-agonist), prazosin (alpha(1)-antagonist), clonidine (alpha(2)-agonist), yohimbine (alpha(2)-antagonist), prenalterol (beta(1)-agonist), terbutaline (beta(2)-agonist), and propranolol (beta(1)- and beta(2)-antagonist). Blood flow in normal skin was reduced by phenylephrine (p<0.001), clonidine (p<0.001) and propranolol (p<0.01), and increased by prazosin (p<0.05), yohimbine (p<0.05), prenalterol (p<0.05) and terbutaline (p<0.01). In partial-thickness burns, blood flow was reduced by phenylephrine (p<0.01), clonidine (p<0.01) and propranolol (p<0.05). In full-thickness burns, only clonidine reduced perfusion (p<0.05). In conclusion, beta(1)- and beta(2)-adrenoceptors play important role in the physiological regulation of skin perfusion but are of lesser importance for postburn skin perfusion. Vasoconstrictive alpha(1)- and alpha(2)-adrenoceptors were shown to be tonically active in normal skin and in partial-thickness burns, exerting a negative effect on skin perfusion which was further potentiated by exogenous administration of alpha(1)- and alpha(2)-agonists and reversed by selective alpha-blockers. In full-thickness burns, activation of alpha(2)-receptors was shown to significantly impair skin circulation, raising a flag of warning for the use of clonidine to treat pain in burn patients.

Role of histamine receptors in the regulation of edema and circulation postburn.

Despite histamine being a potent endogenous vasoactive agent released in increasing amounts postburn, its role in postburn oedema formation has been controversial and its effect on burn circulation poorly investigated. The present study investigated the involvement of H(1), H(2) and H(3) receptors in postburn edema in rats exposed to skin and muscle burns and their influence on skin circulation postburn. We used the selective antagonists clemastine (H(1)), ranitidine (H(2)), thioperamide (H(3)) and the selective H(3) receptor agonist, imetit. Results showed that none of the antagonists or the H(3) agonist had significant effect on postburn edema measured by quantitative spectrophotometric analysis of extravasated Evans blue-albumin in the full-thickness burned skin or muscle. Clemastine and thioperamide failed to induce significant effect on blood flow in the partial- or full-thickness skin burn injury as measured by laser Doppler flowmetry, while ranitidine significantly (P<0.01) reduced blood flow in the full-thickness burn. In contrast, the H(3) receptor agonist, imetit, significantly increased blood flow, both in the partial-thickness burn injury (P<0.05) and in the full-thickness burn (P<0.01). Moreover, imetit significantly (P<0.01) increased mean arterial pressure while thioperamide significantly (P<0.01) reduced systemic pressure. In conclusion, H(1), H(2) and H(3) receptors are not important actors in the regulation of vascular patency permeability, whereas H(3) receptors play an important role by increasing skin circulation postburn, presumably by relaxation of vascular smooth muscle and/or by interacting with other inflammatory neurotransmitters. Data also suggest that H(2) receptor blockers may not be best choice for stress ulcer prophylaxis in burn patients.

The discotheque fire in Gothenburg 1998. A tragedy among teenagers.

The fire disaster in Gothenburg, Sweden, 1998 killing 63 and wounding 213 teenagers was caused by arson committed by a youth from the same community. The fire was started in the basement of an overcrowded discotheque and made, due to unfortunate circumstances, devastating progress. The ensuing rescue work performed by other youth, fire fighters, police and medical staff was prompt and must be seen in the light of a very difficult situation. As a result of these orchestrated efforts and the fact that this disaster occurred in a major city with substantial resources, all the injured were able to be hospitalized within 2h. The load on four local hospitals was initially severe due to the large number of injured and the limited number of staff on night duty. The situation was contained by relocating patients from the intensive care units to ordinary wards and by transporting several of the most severe burn injuries by helicopter to burn units in other parts of Sweden and to Norway. Hundreds of relatives and friends gathered at the local hospitals. This was a new experience for the hospitals and staff, involving many positive aspects as well as some negative aspects such as violence, threats and rumors. As a result of the large number of injuries vast psychosocial rehabilitation program was initiated by health care staff, religious communions, schools and the community, has continued over the past years. Such a disaster emphasises a requirement for extensive preparation not only in the rescue and medical services, but also in the ways and areas to rehabilitate patients in society.

Influence of local anaesthetics on inflammatory activity postburn.

Most studies investigating the pathophysiological processes taking place inside an experimental burn wound use in vitro techniques, which only allow for fragmented measurements of the actual and complex processes occurring inside a burn wound in vivo. In the present study, which used a recently developed in vivo technique in the rat, a full-thickness burn was induced and resulted in the formation of a subcutaneous gelatinous edema with distinct borders to the surrounding connective tissue and free communication with the systemic circulation allowing it to be easily separated for further analysis. In the present study, we investigated the effects of topical local anaesthetics (EMLA) on the inflammatory cascade of a burn wound in vivo. Results showed significantly higher myeloperoxidase (MPO) levels in EMLA-treated burned animals (P<0.01) versus placebo-treated burned controls. EMLA treatment induced a significant inhibition of the synthesis of leukotrien B(4) (LTB(4)) (P<0.001), prostaglandin E(1) (PGE(1)) (P<0.001), prostaglandin E(2) (PGE(2)) (P<0.001) and thromboxane B(2) (TXB(2)) (P<0.001) versus control, while free radical formation did not differ significantly between EMLA-treated and control animals. In conclusion, topical local anaesthetics significantly inhibit the release of several mediators known to take important part in the pathophysiological events ensuing a burn injury, such as activation of pain mechanisms (PGE), oedema formation (LTB), and postburn ischemia (TXB). The increased numbers of leukocytes (MPO) in the burn wound induced by topical local anaesthetic treatment could suggest increased influx and/or increased viability of leukocytes postburn.