u75412e, a lazaroid, prevents progressive burn

American journal of Pathology, Vol. 142, No. 2, February 1993

Copyright ©) American Society for Investigative Pathology

U75412E, a Lazaroid, Prevents Progressive BurnIschemia in a Rat Burn Model

Mihye Choi and H. Paul EhrlichFrom the Wound Healing Laboratory, Shinners BurnsInstitute, Massachusetts General Hospital, Harvard MedicalSchool, Boston, Massachusetts

Thermal energy causes an immediate, irrevers-ible injury at the burn site,folowed by a delayed,reversible tissue loss in the area surrounding theburn site due to progressive ischemia. We inves-tigated the role of lipid peroxidation in thepathogenesis of progressive ischemia in a ratburn modeL The burn model consisted ofa row offour 10 x 20 mm burns separated by threeunburned 5 x 20 mm skin bridges (interspaces).The interspaces became ischemic and necrotic by24 hours, producing a single wound with themerger ofthe burn sites. U75412E, a lipidperox-idation inhibitor, preserved vascular patency,restored blood flow, prevented a rise in tissueconjugated dienes, and maintained tissue viabil-ity in the interspaces. Four separate burnwounds healed between three viable strips ofhair-bearing interspaces. The treatment waseffective, when given systemically during theperiod between 2 hours before and I hour afterthe burn. U75412E prevented progressive burnischemia and the expansion oftissue loss. (AmJPathol 1993, 142:519-528)

Acute tissue destruction occurs at the site of burninjury because of coagulative necrosis caused bythermal energy. In addition, a delayed loss of tissueoccurs in the surrounding, uninjured skin as a con-sequence of progressive ischemia. In 1953, Jacksondescribed three discrete affected areas in burnedskin.1 The zone of coagulation is the directly burnedarea that is irreversibly damaged. The zone of stasisthat surrounds the zone of coagulation is initially per-fused, but blood flow ceases by 24 hours. The zoneof stasis is characterized by the presence of pete-chial hemorrhage and vascular thrombosis at 24hours. Tissue in the zone of stasis is renderedischemic and eventually necrotic. The outer most

affected area surrounding the burn is the zone ofhyperemia. This area always survives.

It was previously postulated that oxygen radicalscontribute to local tissue damage in burn injury.24 Ithas been proposed that the oxygenation products ofthe arachidonic acid cascade contribute to burnedema.3 Ibuprofen has been shown to decreaseburn edema and block the production ofprostanoids.4 It has been demonstrated thatincreased vascular permeability in thermal injury cor-relates with increased xanthine oxidase activity.5 Thexantine oxidase inhibitor, allopurinol, attenuated localburn edema. Other antioxidants such as catalase,superoxide dismutase, and iron chelators are alsoeffective. The mechanism by which oxygen radicalscause local tissue damage has not beendemonstrated. Although it has been postulated thatoxygen radicals contribute to progressive ischemiaand necrosis in the zone of stasis, it has not beenproven.2We hypothesized that, in the zone of stasis, oxy-

gen radicals cause endothelial cell membrane dam-age by lipid peroxidation of the membrane fattyacids. This membrane injury would lead to endothe-lial cell disruption, increased vascular permeability,and local tissue edema. The endothelial cell disrup-tion would, in turn, trigger vascular thrombosis andocclusion, resulting in tissue ischemia and necrosis.Lipid peroxidation products, such as malondialde-hyde (MDA) and conjugated dienes, have beenfound to be elevated in burned skin and serum.67The level of conjugated dienes has been shown tocorrelate with secondary lung injury associated withburns.76 However, a casual relationship between

Supported in part by NIH grant GM-32705, Shriners Hospital ofNorth America, and The Upjohn Company.

Part of this work was presented at the American Burn AssociationMeeting, Baltimore, April 1991 and appeared in abstract form (ABA1991:132).Accepted for publication July 24, 1992.Dr. Choi's present address is Department of Plastic Surgery, Mt.

Sinai Hospital, New York, NY 10029.Address reprint requests to Dr. H. Paul Ehrlich, Shriners Burns

Institute, One Kendall Square, Bldg. 1400, Cambridge, MA 02142.

519

520 Choi and EhrlichAJP February 1993, Vol. 142, No. 2

lipid peroxidation and tissue damage in burn injuryhas not been established.

To test whether lipid peroxidation plays a role inthe development of progressive ischemia in the zoneof stasis, we used U75412E (Figure 1), a lipid per-oxidation inhibitor, in an animal burn model.U75412E is one of a family of molecules known as

lazaroids and is composed of a steroid moiety joinedto an iron-chelating group. The steroid moiety is lipo-philic and integrates into the cell membrane. Itblocks lipid peroxidation with its iron-chelatinggroup9 and scavenges lipid peroxyl radicals in a

manner similar to that of vitamin E.10The rat burn model used facilitates the monitoring

of vascular perfusion, the measurement of lipid per-oxidation, and the assessment of vascular patency.Progressive ischemia and necrosis in the zone ofcoagulation, as well as in the zone of stasis, can beassessed. A brass probe with four prongs separatedby three 5-mm notches produces four distinctiveburn sites separated by three "interspaces" ofunburned skin (Figure 2). The interspaces are notdirectly injured, but within hours of burn injury theyundergo progressive ischemia and at 24 hours theyare necrotic with petechial hemorrhage.11 The inter-spaces represent the zone of stasis and the burnsites represent the zone of coagulation. The combburn allows continuous visual inspection and bloodflow monitoring with a Laser Doppler Perfusion Scan-ner of both the burn sites and the interspaces. Vas-cular patency is assessed by latex vascular castsand histology. The level of conjugated dienes, a

product of lipid peroxidation, was measured from thehomogenates of tissues from the interspaces and theburn sites. These parameters were measured in laz-aroid-treated rats and in untreated control rats.

Materials and MethodsCompoundsU75412E was obtained from The Upjohn Company(Kalamazoo, Ml). It was dissolved in 0.02 mol/L cit-rate buffer (pH 3) with 2.65% glucose at a concen-

tration of 1.5 mg/ml.

Nf

N N

Figure 1. U75N

0

Figure 1. U75412E (MW-644.82).

Site

5k

Inter%paceFigure 2. Comb burn: a brass probe (20 X 20 X 55 mm) with fourprongs separated by three notches was equilibrated in boiling waterand rested on the backs of rats. This resulted in four directly burnedareas (burn sites) separated by tbree uninjured skin bridges(interspaces). 7Te burn sites correspond to the zone ofcoagulation; theinterspaces, the zone ofstasis; and the surrounding area, the zone ofbyperemia.

Thermal InjuryEighty four male Sprague-Dawley rats (300-325 g)were kept under approved standard laboratory con-ditions with veterinary and nutritional support. Theback of each rat was shaved and depilated with Nair(Carter Products, NY). The next day, the brass combprobe equilibrated in boiling water was placed with-out pressure for 30 seconds on one side of the backof a rat under anesthesia (pentobarbital, 40 mg/kg).After reequilibrating the brass probe, a second combburn was made on the other side of the back. Thedimensions of the brass comb were 20 x 20 x 55mm with four 10 x 20 mm rectangular prongs, sep-arated by three 5-mm wide grooves. Application ofthis heated brass comb resulted in four rectangularburns of 10 x 20 mm separated by three 5 x 20 mmunburned areas (interspaces) (Figure 2).

Blood FlowBlood flow in the burn sites or the interspaces wasmeasured with a Laser Doppler Perfusion Scanner(Med Pacific LD 5000, Seattle, WA). Measurementswere made pre-burn and at 1-, 2-, 3-, 4-, 8-, 16-, and24-hour intervals post-burn. The measurements wereexpressed in millivolts and averaged from the burnsites and the interspaces separately for each timepoint. These averaged values were compared withthe pre-burn skin values from each animal and theresults were expressed as percentage of normal skinblood flow.

Vascular CastsVascular casts were made at 24 hours. Two rats fromeach group in experiment 1 were anesthetized withpentobarbital and open thoracotomy was performed

Lazaroid's Prevention of Burn Ischemia 521AJP February 1993, Vol. 142, No. 2

to expose the ascending aorta. Yellow latex com-pound (Microfil, Canton Bio-medical Products, Boul-der, CO) was injected into the ascending aorta afterthe entire vascular tree was flushed with 10%formalin. The comb burn with 1 cm of surroundinguninjured skin was excised and dehydrated in aseries of ascending concentrations of ethanol over 5days. The dehydrated preparation was immersed inmethylsalicylate to render the tissue transparent.

HistologyThe vascular casts were rehydrated and a strip ofskin (3 x 15 mm) was excised from the center ofeach comb burn. This skin strip included the inter-space at the center and the burn sites at the edges.The skin samples were embedded, sectioned, andstained with hematoxylin and eosin. These sectionswere examined by light microscopy.

Wound ContractionThe photographs of three rats from each group inexperiment 1 were taken on the day of injury and 5days after. The burn areas were cut out from thephotographs and weighed. The changes in weightsover 5 days for the lazaroid-treated group and theuntreated group were compared using the followingformula as a consequence of wound contraction:

Wound Contraction (%) =

1 - Weight of Cut-Out at Day 5Weight of Cut-Out at Day x 100 (1)

Lipid Peroxidation Product (ConjugatedDienes)Lipid peroxidation was assessed by measuring con-jugated dienes which are lipid peroxidationproducts. The conjugated dienes were chloroform-extracted from skin and their ultraviolet absorptionwas measured.12 Briefly, 0.8 ml of distilled water wasadded per 1 g of wet tissue and the tissue wasprocessed in a Brinkman homogenizer for 1 minute.Six milliliters of a 1:2 mixture of methanol-chloroformpreheated at 45 C was added to the homogenateand vortexed for 2 minutes. Two milliliters of chloro-form was added and vortexed for 30 seconds. Next,2.0 ml of acidic water (pH 2.5, adjusted with 1 N HCI)was added and vortexed for 30 seconds. The mix-ture was centrifuged at 1000 x g for 10 minutes at 20C. Two milliliters of the chloroform layer was aspi-rated and dried under a stream of nitrogen gas at 37C. The residue was resuspended in 1.0 ml of hep-tane and analyzed for ultraviolet absorption by a

spectrophotometer at 233 nm. Findings werereported in optical density units per gram of tissue.

Experimental DesignExperiment 1: The Effect of LazaroidTreatmentTwenty-one rats were randomized into three groupsand the comb burns were administered. Immediatelyafter the burn, the first group received a 2 mg/kginjection of U75412E intramuscularly. The secondgroup received an equal volume of vehicle-alone inthe same location. The third group received noinjections. Blood flow was monitored by the LaserDoppler Perfusion Scanner. Two rats from eachgroup were killed at 24 hours for latex vascular castsand histology. The remaining five rats from eachgroup were evaluated at 5 days for woundcontraction.

Experiment 2: Treatment WindowThirty rats were randomized into six treatmentgroups. The rats in each group received single injec-tions of U7541 2E (2 mg/kg intramuscularly) at 1, 2, or3 hours before the comb burn injury and 0, 1, or 2hours after the burn injury. Blood flow was monitoredwith the Laser Doppler Perfusion Scanner pre-burn,and at 1, 2, 3, 4, and 24 hours post-burn. All ratswere killed at 24 hours.

Experiment 3: Lipid Peroxidation Product(Conjugated Dienes)Eighteen rats were randomized into two groups.Immediately after the comb burns were inducedbilaterally, the first group received a 2 mg/kg dose ofU75412E intramuscularly and the second groupreceived no injections. Normal skin was biopsiedfrom each rat before the burn injury as control. Threesets of comb burns were excised for each time pointat 15 and 30 minutes and 1, 2, 3, and 4 hours post-burn for both groups. The burn sites and the inter-spaces of each comb burn were removedseparately. The tissues were immediately frozen inliquid nitrogen and stored at -70 C untilhomogenization. At the time of homogenization, theunderlying subcutaneous fat and muscles weretrimmed from the samples and only the skin wasprocessed for measurement of conjugated dienes.

StatisticsThe blood flow levels, the degrees of wound contrac-tion and the concentrations of conjugated dienes


were expressed in means plus or minus standarddeviations. The unpaired two-tailed Student's t-testwas used to compare the results among the control,the vehicle-alone, and the lazaroid-treated groups.The significance was attributed to P < 0.05.

Results

No statistical differences were noted in the bloodflows of the burn sites in the 1- to 24-hour time pointsafter the burn injury among all groups by Laser Dop-pler Perfusion Scanner monitoring. Blood flow levelsin the burn sites in all three groups registered 6% ±2% to 13% ± 5% of the pre-burn blood flow levels.The blood flow levels of the interspaces were notsignificantly different in the 1- to 4-hour time points.At 8 hours, the blood flow in the interspaces of thelazaroid-treated rats was 36 ± 12% of the pre-burnblood flow level, which was higher than but not sta-tistically different from the blood flow levels (23 ± 9%and 21 ± 4%) in the interspaces of the vehicle-aloneand the control groups, respectively. At 16 hours, theblood flow in the lazaroid-treated interspaces rose to75 ± 29%, whereas the blood flows in the vehicle-alone and the control interspaces remained dimin-ished (25 ± 7% and 29 ± 10%) (P < 0.02). At 24hours, the interspaces of the lazaroid-treated groupshowed a 152 ± 26% increase in blood flow, com-pared with 45 ± 7% and 35 ± 9% for the vehicle-alone and the control groups (P< 0.001). There wereno differences in the burn site blood flows, whichshowed 11 ± 2%, 10 ± 3% and 10 ± 3% for therespective groups at 24 hours (Figure 3).The gross appearances of the burn sites were the

same for all three groups. The vehicle-alone and thecontrol interspaces developed small petechiae by 4hours and showed signs of necrosis by 24 hours.However, 26 of 30 interspaces of the five lazaroid-treated rats failed to develop small petechiae by 4hours and displayed hyperemia at 24 hours. Four of30 interspaces located at the caudal end in the laz-aroid-treated rats showed some signs of petechiaeat 4 hours and necrosis at 24 hours (Figure 4). Thismay be attributed to the convexity of the torso of ratswith the brass probe resting heavier just above theiliac crest which is the lowest point of the torso. Intotal, four of 30 interspaces in the lazaroid-treatedrats showed necrosis compared with 60 of 60 inter-spaces in the placebo and the control rats. There-fore, treating rats with the lazaroid intramuscularlyimmediately after the burn reliably produced the sur-vival of the interspaces in 26 of 30 interspaces at 24hours. Lazaroid treatment decreased the area of tis-

200 -

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00 8 16 24

HoursFigure 3. Blood flow changes: blood flow in the burn sites and theinterspaces wasfollowed for 24 hours with a Laser Doppler PerfusionScanner. Up to 4 hours there was no significant differences between theuntreated, vehicle-alotne, and the lazaroid-treated groups. Between 8and 24 hours, the bloodfloul in the interspaces ofthe lazaroid-treatedrats (solid triangle) increased. At 24 hours, the interspaces of thelazaroid-treated rats showed a threefold increase in bloodflow com-pared with the vehicle-alonie group (solid square) (P < 0.001). Nosignzificant difference was noted between the burn sites of the vehicle-alone (open sqtare) antd the lazaroid-treated (open triangle) groups.

sue loss by 26% as a consequence of the survival ofthe interspaces located between the burn sites.

At 5 days, the vehicle-alone and the control combburns showed complete necrosis of the interspacesforming an eschar continuous with the burn sites.However, the lazaroid-treated comb burns showedviable interspaces separating four eschar-coveredburn sites. The interspaces also showed hair growthindicating surviving hair follicles. The four of 30 inter-spaces that showed necrosis at 24 hours were notviable at 5 days (Figure 5). The appearance of theinterspaces at 24 hours was a reliable indicator forthe eventual outcome at 5 days.

The latex vascular casts made at 24 hoursrevealed thrombosed blood vessels in both the burnsites and the interspaces of the vehicle-alone andcontrol groups. The latex casts of the untreated ratscontained single, large, rectangular defects. In thelazaroid-treated group, the interspaces containedpatent blood vessels, but no patent blood vesselswere found within the burn sites. The comb burns ofthe lazaroid-treated rats were composed of four sep-arate rectangular defects (Figure 6).

The rehydrated vascular casts were biopsied forhistology instead of fresh skin because latex isclearly identifiable by light microscopy as blackintraluminal constituent and latex-filled blood vesselsrepresent patent vessels at the time of latex injection.The histology of all burn sites showed coagulativenecrosis to the panniculus carnosus. No latex-filledvessels were identified in the dermis of the burn

-


A

R

Figure 4. Comb burnis at 24 honis:. the interspaces of the control anldthe vehicle-alone rats (A atnd B) u'ere necrotic uwitbin 24 hoirs. Tuentysix ojf 3O interspaces ofthefive lazaroid-treated rats were viable (870o).Fotur interspaces at the candal-ends. shooed somle signs of necrosis inthe lazaroid-treated rats (C). In total, J]tur of 30 intenspaces in thelazaroid-treated rats shouwed necrosis (130%) comtlpared with 60 of 60interpaces in the control and the vehicle-alone rats.

sites, although many latex-filled vessels wereobserved below the panniculus carnosus muscle.The histology of the interspaces of the vehicle-aloneand control groups also revealed the absence oflatex-filled vessels in the dermis. Numerous vesselswere filled with organized thrombi or packed with redblood cells in stasis. In contrast, the interspaces ofthe lazaroid-treated rats showed numerous latex-filled vessels and the absence of thrombosed ves-sels throughout the dermis (Figure 7).

The findings from the time window experimentwere the lazaroid-treated comb burns displayed min-imal wound contraction compared with the controlcomb burns. The average cut-out weight, as a mea-sure of wound area of comb burns on the day of burninjury, was 647 ± 15 mg for the lazaroid-treatedcomb burns and 625 ± 47 mg for the vehicle-aloneand control comb burns. The fifth day, average cut-

Figure 5. Comb buirtns at 5 days: the interspaces ofthe conttrol and thevehicle-alone rats (A anid B) showed complete necrosis andformed a.singlce large continuous escbar with the buirni sites. 7Te interpace.s ofthe lazaroid-treated comnb buirnis appeared healthy bridging fouir sep-arate eschar-covered burn sitcs. Hair grouwth as obsenred ini tbeseinterpaces (C).

out weight of the lazaroid-treated burns was 577 ±52 mg, compared with 515 ± 40 mg for the untreatedcomb burns. The rate of contraction (using the abovedescribed formula) was 1 1 % for the lazaroid-treatedburns and 18% for the vehicle-alone and controlburns. The degree of wound contraction of the vehi-cle-alone and control burns over 5 days was statis-tically significant compared with that of the lazaroid-treated burns (P < 0.01). This reduced rate of woundcontraction with lazaroid treatment is consistent withthe diminished degree of tissue loss.No statistical difference in blood flow was noted

for the burn sites or the interspaces among the sixtreatment groups up to 4 hours post-burn. The blood


i.L _ s

Figure 6. Latex vascular casts: vascular casts are shown from 24hour-old comb burns. 7The lazaroid-treated group (A) maintainedpatent blood vessels in the interspaces. In the control group, the vesselswere occluded because of thrombosis and were void of latex (B).

flow readings at 24 hours were also indistinguishablefor the burn sites of all treatment groups. However, at24 hours, the interspaces of the group that was

treated with the lazaroid at 1 hour before, 0 hour, or

1 hour after burn injury showed hyperemia andincreased blood flows to 183 ± 7%, 152 ± 27%, and123 ± 21 % (P < 0.001) from the pre-burn blood flowmeasurements. When lazaroid was given 2 hoursbefore burn injury, the interspace blood flow was

restored to a pre-burn level of 90 ± 12%. No hy-peremia appeared in the interspaces when the ratswere pretreated 2 hours before burn injury. The inter-spaces of the groups that received lazaroid 3 hoursbefore and 2 hours after burn injury showed 35 and41% of the pre-burn blood flow levels, which were

not statistically different from the values of the vehi-cle-alone and the control groups in experiment 1.Lazaroid therapy at these times was ineffective atrestoring blood flow to the interspaces. Lazaroidtreatment was found to be effective in preventingprogressive ischemia and necrosis if given duringthe period between 2 hours before and 1 hour afterburn injury (Figure 8).

Conjugated diene concentration in the burn sitesreached a peak at 30 minutes post-burn that was 2.4times that of normal (uninjured) skin (Figure 9). Thelevels of conjugated dienes returned to base line by3 hours. Treatment with U75412E diminished theconjugated diene level in the burn sites significantly(P < 0.01). The peak concentration of conjugateddienes in the lazaroid-treated burn sites was only 1.6times the level of conjugated dienes in uninjuredskin. In addition, the occurrence of the peak levelwas delayed to 1 hour post-burn. The interspaces of

the control rats showed peak concentrations at 15minutes post-burn and this was 3 times the conju-gated diene level in uninjured skin. This peak level ofconjugated dienes remained elevated at 30 minutes,and then returned to the normal level by 2 hours. Theconjugated diene level in the lazaroid-treated inter-spaces never rose more than that of the uninjuredskin. U75412E prevented any rise in conjugateddiene levels in the interspaces of the treated rats andthis was significant compared with the untreated rats(P< 0.01).

Discussion

In the comb burn model, the burn sites represent thezone of coagulation, showing irreversible tissue losswith vascular thrombosis and tissue necrosis. Theinterspaces are equivalent to the zone of stasis. Theinterspaces on the surface of skin are proposed tobe continuous with and representative of the zone ofstasis below the burn sites. If left untreated, this tis-sue dies because of progressive ischemia. Treatingwith the lazaroid did not promote the recovery oftissue in the burn sites (zone of coagulation), butcaused the reversal of tissue loss in the interspaces(zone of stasis).

The most dramatic difference in the lazaroid-treated burns compared with the untreated burns isthe presence of patent dermal vessels in the inter-spaces of the comb burns as demonstrated bothby latex vascular casts and histology. Blood flowmeasured by a Laser Doppler Perfusion Scannerincreases rapidly between 8 and 24 hours. It is pro-posed that this increase in blood flow readings in theinterspaces of U75412E-treated animals does notrepresent shunted blood flow, but the actual perfu-sion of skin through the patent vasculature. Visualinspection of the interspaces of the treated rats dem-onstrates pink and viable skin bridges comparedwith the hemorrhagic and necrotic interspaces of theuntreated rats at 24 hours. In 5 days, these lazaroid-treated rats interspaces appear healthy with hairgrowth. Reduced wound contraction measured inthe lazaroid-treated rats is indicative of the survivinginterspaces. These skin bridges function as internalsplints preventing wound contraction. There is noevidence that lazaroid treatment has an inhibitoryaction on wound contraction.

In the comb burn model, the burn sites are fullthickness burns where all the skin appendages withtheir reservoirs of keratinocytes are destroyed. In asecond-degree burn, the epidermal cells for resur-facing the burn wound are derived from the skin

II


Figure 7. Histologyfrom vascular casts: the representative sections ofa burn site and an itnterspacefrom a control rat and a lazaroid-treated ratare shown. These sections are madefrom the skin biopsy samples of the rehydrated vascuilar casts. The black initraluminal material in blood vesselsis latex. Thercfore, these vessels were patent at the time of latex inijection at 24 hours. The burn sites of both the control (A) and lazaroid (C) groupsshouw the absence of latex-filled, patent vessels and the presence ofthrombosed vessels in the dermis. Note the latex-filled vessels beloun the pantzicuiluiscarnosus muscle in the burn sites, indicating adequate latex-filling. The itnterspace ofthe conitrolgrouip (B) shous thrombosed vessels antd the absenceof latex-filled vessels in the dermis. In contrast, the itnterspace of the lazaroid-treated group (D) contains muiltiple latex-filled vessels throuighouit thedermis (arrous). (Magntificationi X31).

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HoursFigure 8. Blood flou' with various times of therapy: at 24 hours, sig-nificant increases in bloodflou in the interspaces were noted by theLaser Doppler Perfusion Monitor u'hen lazaroid treatment u'as given 1hour before, 0 hour, or 1 hour after burn injury. Bloodflouw levels wereelevated creating hyperemia in the interspaces. The group treated 2hours before burn injury bad bloodflou restored to the preinjury leveland no hyperemia was noted. The groups treated 3 houirs before and2 hours after burn injury did not show any bloodflou' recovery.

appendages. The skin appendages in a deep sec-ond-degree burn would be included in the zone ofstasis and the survival of these appendages wouldbe critical in a clinical setting. The fate of tissue in thezone of stasis in a deep second-degree burn requir-ing skin grafts or heals by epithelialization from thesurviving keratinocytes in the skin appendages. Thebeneficial effect of lazaroid treatment in the inter-spaces of this model implies survival of skin append-ages in the zone of stasis by preventing progressiveischemia.How does U75412E preserve vascular patency

and perfusion in the interspaces? The significantreduction in the level of conjugated dienes in theburn sites and the absence of any elevation in theinterspaces during the immediate post-burn period,support the concept that the beneficial effect seenwith U75412E is due to the inhibition of lipidperoxidation. It is noted that ischemia and necrosiswithin the interspaces were inhibited when the laz-aroid was given 1 hour after the burn injury in thetime window study (experiment 2). According to theconjugated diene measurements, this is after thepeak level of conjugated dienes was reached in theinterspaces. It is puzzling how lazaroid given afterthe peak activity of lipid peroxidation could beeffective. This may be explained by the fact thatconjugated diene measurements do not assess allactivities of U75412E. This compound also scav-enges lipid peroxides,9 which are formed after theconjugated dienes are produced by lipid peroxida-

enwzw

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Figure 9. Skin conjugated dienes. Upperpanel: burn site. Conjugateddienes were measuredfrom tissue homogenates. The conjugated dienelevel ofthe burn sites ofthe control rats (solid triangle) reached apeak30 minutes postburn that was 2.4 times higher than that ofuninjuredskin. The conjugated diene level of the burn sites of the lazaroid-treated rats (open triangle) was diminished significantly at 15 and 30minutes post-burn (P < 0.01). Bottom panel: interspace. The conjui-gated diene level reached a peak 15 minutes post-burn and remainedelevated at 30 minutes in the control rats (solid diamond); however,the interspacesfrom the lazaroid-treated rats (open diamond) did notshow any elevation at any time point (P < 0.001).

tion of unsaturated fatty acids.11 The thiobarbituricacid assay, which measures MDA product of lipidperoxidation, is theoretically a better choice to eval-uate the activity of lazaroid, because MDA is pro-duced after lipid peroxides are formed.11 However,MDA is an unstable metabolite of lipid peroxidation invivo. It is shown in vitro that 98% of the MDA mea-sured was an artifact of heating in the thiobarbituricacid assay.12 Here the conjugated diene assay waschosen for the assessment of lipid peroxidation dueto the stable product and the reproducibility of theassay. It is also noted that although the level of con-jugated dienes in the burn sites is initially diminishedwith lazaroid therapy, this effect is not translated intoany demonstrated improvement in burn site survival.At 1 hour after the burn, the conjugated diene levelsof the treated and the untreated burn sites are ele-vated and indistinguishable from each other. Thismay reflect that the thermal damage in the burn sitesis complete and therefore irreversible.

n


We speculate that the inhibition of lipid peroxida-tion by U75412E preserves vascular patency by pro-tecting endothelial cells. The prevention of endothe-lial cell membrane injury results in preservation ofendothelial cell integrity, thus inhibiting tissueedema, vascular occlusion due to thrombosis, andeventual tissue ischemia and necrosis.One of the compounds in the lazaroid family, Tir-

ilazad has been reported to inhibit lipid peroxidationin cerebral ischemia13 and spinal cord injury.14 Otherpathological conditions such as myocardialischemia and flap ischemia may benefit from theinhibition of lipid peroxidation with a lazaroidcompound. Oxygen radicals are involved in variouspathological conditions15 including cerebral,16 car-diac,17 and renal ischemia,18 as well as in the agingprocess12 and flap ischemia.19,20 the sources of oxy-gen radical production and their harmful effects havebeen documented in literature. Various biochemicalmodalities to prevent this type of injury have beendescribed. Inhibitors of the arachidonic acid cas-cade, such as cyclooxygenase inhibitors, leuko-triene receptor agonists, and thromboxane syn-thetase inhibitors are shown to decrease local burnedema and improve dermal perfusion.352123 Theincrease in xanthine oxidase seems to play a majorrole in burn edema, endothelial cell injury and com-promised microvascular integrity. Allopurinol, a xan-thine oxidase inhibitor, attenuates these changes.5'22There is substantial evidence demonstrating the rela-tionship between secondary lung injury after thermaltrauma of skin and the lipid peroxidation products inplasma and tissue.7,8,24,25 Iron chelators, whichinhibit the Fenton reaction, an initial step in the lipidperoxidation process, are effective in reducing burn-induced lung injury.8 Neutrophil depletion protectsagainst lung injury associated with thermal trauma.26Catalase and superoxide dismutase are found toavert cytotoxic injury to endothelial cells by activatedleukocytes.7 However, lung injury induced by otherinsults, such as endotoxin, did not improve with neu-trophil depletion.27 A monoclonal antibody (MAb60.3) directed to a human neutrophil membrane sur-face glycoprotein, which is responsible for neutrophiladhesion to endothelial cells, was shown to reducemultiple organ injuries associated with hemorrhagicshock and reperfusion injury.28-30 A recent studyshowed that MAb 60.3 also ameliorated burn woundrepair by the enhancement of epithelialization in sec-ond-degree burns in a rabbit model.31

In conclusion, this study demonstrates thatU75412E, a lipid peroxidation inhibitor, averts pro-gressive burn ischemia. It is suggested that the laz-aroid effect is related to the preservation of the integ-

rity of the endothelial cell membrane and therestoration of vascular patency in the zone of stasisby inhibiting iron-dependent lipid peroxidation reac-tion and scavenging lipid peroxides. U75412E waseffective in preventing progressive burn ischemiawhen rats were treated between 2 hours before to 1hour after burn injury. The levels of conjugateddienes were significantly diminished in the burn sitesand the interspaces in the lazaroid-treated rats.

AcknowledgmentsThe authors wish to thank Blanca Lusetti and JosephRajaratnam for technical assistance.

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u75412e, a lazaroid, prevents progressive burn

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