T H E E F F E C T O F S E X S T E R O I D S O N T H E A O R T I C E N D O T H E L I U M O F R A T S W I T H T H E R M A L I N J U R Y Ş u le C e tin e l, M . D . * / E m el S. Ö z v e r i, M .D .* * / A y h a n B o z k u r t, P h .D .* * * G o n c a g ü l H a k la r , M . D . * * * * / T a n g ü l Ş a n , P h .D .* / B e r r a k Ç. Y e ğ e n , M .D .* * * * D e p a r t m e n t o f H i s t o l o g y a n d E m b r y o l o g y , S c h o o l o f M e d i c i n e , M a r m a r a U n i v e r s i t y , I s t a n b u l , T u r k e y . ** D e p a r t m e n t o f G e n e r a l S u r g e r y , S c h o o l o f M e d i c i n e , M a r m a r a U n i v e r s i t y , I s t a n b u l , T u r k e y . * * * D e p a r t m e n t o f P h y s i o l o g y , S c h o o l o f M e d i c i n e , M a r m a r a U n i v e r s i t y , I s t a n b u l , T u r k e y . ' * ' * D e p a r t m e n t o f B i o c h e m i s t r y , S c h o o l o f M e d i c i n e , M a r m a r a U n i v e r s i t y , I s t a n b u l , T u r k e y . ABSTRACT
O b jective: Thermal injury-induced physiopathological events are related to an acute inflammatory reaction. This study was designed to observe the role of sex steroids on the aortic endothelia of rats with burn injury.
M ethods: Male Wistar Albino rats were given burn trauma (n=30), and the rats were injected subcutaneously with either 17(3 estradiol benzoate (E2, 10mg/kg) or an androgen receptor blocker cyproterone acetate (CPA, 25 mg/kg) or vehicle immediately after the burn and at 12 h. At the 24th hour of burn injury, the rats were decapitated. Blood samples to measure serum TNF levels were collected. The aortas of the rats were processed for both light and scanning electron microscopy. Student’s t test was used for statistical analysis.
R esults: Antiandrogen and E2 treatments depressed burn induced elevation in serum TNF levels. Both light and electron microscopy revealed a morphology in accordance with TNF results. The endothelia of aortas were preserved significantly better than in the vehicle group. The
adhesion of blood cells on endothelium was reduced in the antiandrogen or E2 - treated groups.
Conclusion: In conclusion, we may predict that
treatment with estrogens or antiandrogens may have an ameliorating effect on systemic inflammation induced by burn.
K e y W o rd s : Burn injury, Sex steroids, Aorta, Microscopy
IN T R O D U C T IO N
Burn-induced injury produces local and systemic damage due to the release of several inflammatory mediators. The physiopathological events after thermal injury are related to an acute systemic inflammatory reaction (1). Shortly after thermal injury, a number of cytokines (interleukin- 1, TNF) are induced and inflammatory response develops (1). Overproduction of cytokines and the activation of endothelial cells lead to production of inflammatory active substances. TNF is among the most important cytokines
M a r m a r a M e d i c a l J o u r n a l 2 0 0 3 ; 1 6 ( 2 ) : 9 7 - 1 02
Correspondence to: Şule Çetine!. M. D, - Department of Histology and Embryology, School of Medicine, Marmara University, Haydarpaşa, 34668, İstanbul
involved in the acute inflammatory reaction following thermal injury (2,3). It enhances the endothelial adhesiveness of leukocytes and stimulates neutrophils and monocytes promoting their adherence, phagocytosis and degranulation (1). Burn injury leads to vascular thrombosis and occlusion by thermal damage to the vascular network and may cause changes in the
endothelium. The resulting response is
vasoconstriction due to the release of ET-1 and a reduction in vasodilator PGI2/ NO (4, 5) response.
Animals with thermal injury showed significant loss of systemic vascular resistance and fall in mean arterial pressure (6,7). Meanwhile, alteration in vascular smooth muscle receptor activity has been hypothesized to occur (8). Also maximal rise of the spontaneous intravascular platelet aggregation index was observed in studies on burn injury (9). The frequency of thromboembolic complication in burn patients has been shown to range from 0.4 -7 % (1, 4). Leukocytes and the process of leukocyte adherence have been implicated in the pathogenesis of organ dysfunction after thermal injury (8, 10).
Recent clinical studies in trauma patients have verified that susceptibility to and death from sepsis is higher in men than in women (11-13). Female animals demonstrated normal or enhanced immune response as opposed to markedly decreased immune response in male mice in recent clinical studies (11-13). Besides, the suppressive effect of androgens on immunity have been reported on normal immune functions as well as in autoimmune diseases (14,15). However, it remains unclear whether sex steroids have any effect on vascular integrity challenged with thermal injury. So the aim of this study was to investigate the role of endogeneous testesterone or exogenous estradiol on the aortic endothelia of rats with burn injury.
M A TER IA LS A N D M E T H O D S
Animals: Male Wistar Albino rats (250-300g) were kept in a light and temperature-controlled room on a 12:12-h light-dark cycle, where the temperature (22±0.5 °C) and relative humidity (65-70 %) were kept constant. They were fed a
standard diet and water ad libitum. The experiments were approved by the Marmara University, School of Medicine, Animal Care and Use Committee. Surgical procedures and burn trauma were conducted under anesthesia performed by intraperitoneal (ip) injection of a
mixture of ketamine (100mg/kg) and
chlorpromazine (12.5mg/kg).
Burn injury. Under anesthesia, the dorsum of
the rat was shaved and exposed to 90 °C water bath for 8 seconds, to induce full-thickness burn involving 30% of the total body surface area. Fluid resuscitation was made (20 ml/kg saline; subcutaneously, sc) and the animals were placed on a heating pad until they recovered from anesthesia.
Experim ental design. The rats were treated sc twice- immediately after burn and at 12 h- with either 17(3 estradiol benzoate (10 mg/kg, in olive oil; Sigma) or an androgen receptor blocker cyproterone acetate (CPA, 25 mg/kg; Schering A.G.) or oil alone (vehicle, 1 mg/kg; Komili) before they were decapitated at the 24th hour. Trunk blood obtained by decapitation was taken into tubes, which were kept on crushed ice. Serum samples were kept at -50 °C until TNF levels were assayed.
Measurem ent of TN Fu levels. A TNFrx enzyme-
linked immunabsorbent assay kit (Endogen, Mass, USA) was used with the manufacturer’s instructions without any modifications. Optical densities were read on a plate reader set at 450 nm. The concentration of TNFa in the serum samples was calculated from the standard curve, multiplied by the dilution factor and expressed as pg /mL.
Light m icroscopy, (LM). The rat aortas were
fixed in 10% buffered formalin and dehydrated in increasing concentrations of ethanol, then cleared in toluene and embedded in paraffin. Sections 5 pm thick were stained with toluidin blue (TB) and observed under an Olympus photomicroscope (Tokyo, Japan).
S canning e le c tro n m ic ro s c o p y , (SEM). Specimens were fixed, dehydrated and dried with liquid C 0 2 under pressure with critical point dryer (Bio-Rad E 3000, Hertfordshire, UK), then coated with gold approximately 400 A in a sputter
Sex steroids on thermal injury-induced aortic endothelium
coating unit (Bio-Rad SC 502, Hertfordshire, UK). Coated specimens were veiwed in a Jeol JMS SEM (Tokyo, Japan).
Statistical analysis. All data are expressed as means ± SEM with eight to ten rats per group. Instat statistical package (GraphPad Software, San Diego, CA, USA) was used. Following the assurance of normal distribution of data, one-way analysis of variance (ANOVA) with the Tukey-Kramer post-hoc test was used for multiple comparison. Values of p<0.05 were regarded as significant.
RESULTS
L ig h t m ic r o s c o p y : The control aorta
demonstrated regular endothelium with intact intima layer containing elastic fibres (Fig. 1a). In
the vehicle-treated animals with burn injury, aorta endothelium was disorganized and muscle cell contraction was present (Fig. 2a). In the
estradiol-treated group, almost regular
endothelium and elastic fibers were observed (Fig. 3a). As a result of CPA treatment, the muscle bundles among the elastic laminae showed a contracted morphology, but the luminal contour was regular (Fig. 4a).
S c a n n in g e le c t r o n m ic r o s c o p y : In the control
group, the luminal surface of the aortas was characterized with intact longitudinal relief of elongated and orderly populated endothelial cells with a few blood cells sticking to the luminal surface (Fig. 1b). In the vehicle group, burn injury resulted in a disorganized surface appearance, and numerous blood cells stuck to the lumen were prominent (Fig. 2b). In the estradiol-treated group, the luminal surface
F i g . l : C o ntrol a orta a) re g u la r e n d oth e liu m w ith intact intim a layer (arrow ) co ntaining e la stic fibres, TB X100, b) lum inal surface of aortas was characterized with Intact lo n gitudinal relie f of elongated and orderly populated end oth e lia l cells (arrows) with a few blood ce lls stickin g to the lum inal surface, SEM.
F i g . 2 : Vehicle-treated group, a) aorta endothelium was disorganized (arrow) and muscle cell contraction was present TB X100, b) a disorganized surface appearance, and numerous blood cells (arrow) stuck to the lumen were prominent, SEM.
exhibited nearly no detectable surface changes as compared to the control group, and decreased blood cell adhesion was observed in this group (Fig. 3b) . In the CPA- treated group, the acanthotic eyhrocytes were partially stuck to the luminal surface, which showed almost characteristic regular cobblestone appearance. (Fig. 4b).
S e r u m T N F le v e ls : In the vehicle-treated burn
group (44.03±6.90 pg/ml; p<0.05), serum TNF level was significantly increased compared to the control group ( 25.55±1.92 pg/ml; p<0.05) (Fig. 5). This burn-induced rise in serum TNF level was abolished by treatment with estradiol (27.54 ±3.45 pg/ml; p<0.05) or CPA (29.06±5.72 pg/ml; p<0.05).
F l g .3 : Estradiol-treated group, a) almost regular endothelium and elastic fibers were observed TB X100, b) luminal surface exhibited nearly no detectable surface changes as compared to control group. SEM.
F i g . 4 : CPA treatment, a) the muscle bundles among the elastic laminae showed a contracted morphology, but the luminal contour was regular TB X100, b) the eyhrocytes (arrows) were partially stuck to the luminal surface, which showed almost characteristic regular cobblestone appearance, SEM. □ Control □ vehicle treated 0 E2-treated H CPA-treated 1 4 5 j O BURN
-F i g .5 s Serum TNF levels in the control, vehicle, estrogen and CPA treated rats.
* p<0.05, compared to control group, + p<0.05, compared to vehicle-treated group.
Sex steroids on thermal injury-induced aortic endothelium
D IS C U S S IO N
In the present study, CPA and estradiol treatment did not cause any significant changes in the morphology of the luminal surface of burn-injured aorta endothelium. However, there was a significant decrease in the number of adhering blood cells when compared to vehicle group where the regular topography of the endotelial layer was disrupted. The presence of estradiol prevented the burn-induced morphological changes indicating endothelial damage. In concert with estradiol, CPA also seems to exert an ameliorating effect on luminal morphology of the aorta endothelium.
The model of a 30% total body surface area burn injury results in hypotension and hypoxemia in the burn group (10). Burned animals show a sustained 20-30% fall in mean arterial blood pressure (7). The frequency of thromboembolic complications in burn patients has been estimated to range from 0.4% to 7% (4). Thermal injury leads to activation of strong cytokines. Platelets are inflammatory cells (16); when the endothelium is damaged, platelets adhere to it, thereby becoming activated. Activated platelets release a number of substances such as serotonin, PAF, TNFa, cationic proteins and proteolitic enzymes (collagenase and elastase) that modify tissue integrity (9). Most possibly the antiinflammatory effects of estrogen on burn injury involve mechanisms that reduce both neutrophil infiltration and TNFa which have leading roles in the inflammatory processes. Hence, it is clarified that in the presence of estradiol less platelets adhere to the luminal surface of aorta endothelium.
Prostacyclin and the endothelium derived nitric oxide (NO) are produced by endothelium and have leading roles in managing vascular tone. Studies have shown that NO precursor L- arginine and the NO-donor compound FK 409 which inhibit platelet aggregation and adhesion in the vascular endothelium, lead to an inhibition in balloon injury-induced intimal hyperplasia (17). Moreover, in some studies it is suggested that the ameliorating effect of estrogen may be mediated by the stimulation of prostaglandin synthesis by the vessel wall (17,18).
In the development of cardiovascular disease, the gender difference has been documented in human and animal studies (19,20). Many studies revealed that estrogen might have protective effects on cardiovascular morbitidy (17,18,21). The increased risk of coronary heart disease in young women with bilateral ovariectomy and the beneficial effect of estrogen replacement therapy in postmenaposed women further support a role
for estrogen in protecting against the
development of coronary artery disease (22). It is suggested that estrogens show their effect through a direct action on blood vessels which contain specific high affinity receptors for estrogen (17).
Many studies predict that androgenic steroids
may lead to vasospasm, platelet
hyperaggregability and thrombotic
cardiovascular diseases (19,23). In experimental studies of arterial thrombosis and platelet aggregability models, gender differences have been observed and also an increase in both arterial thrombus formation and platelet aggregability is observed by testesterone
pretreatment (23). It is predicted that
testesterone receptor blockade inhibits the vasoconstrictive activity of thromboxane A2 and/or enhances the release of vasodilator prostacyclin in the aortic-coronary circulation, causing a better organ perfusion after trauma (23).
Our study demonstrated that both estrogen and antiandrogen treatments which lead to an increase in plasma estradiol levels (24) lead to a decrease in burn-induced inflammation in the endothelia of aortas. The pronounced effects of these treatments may be, in part, due to the limiting effect of estrogen on tissue injury by depressing neutrophil infiltration and by reducing the release of inflammatory cytokines. In conclusion, we may predict that treatment with estrogens or antiandrogens may ameliorate systemic inflammation induced by burn.
REFERENCES
1. D e v e c i M , E s k i M, $ e n g e z e r M , t \ is a U. E f f e c t s o f c e r i u m n i t r a t e b a t h in g a n d p r o m p b u r n w o u n d e x c is io n o n IL - 6 a n d T f l f '- a le v e ls In b u r n e d a n im a ls . B u r n s 2 0 0 0 : 2 6 : 4 1 - 4 5 .
2 . S p a r k e s B G . I m m u n o l o g i c a l r e s p o n s e s t o t h e r m a l in ju r y . B u r n s 1 9 9 7 ; 2 3 : 1 0 6 - 1 ¡ 3 . 3 . A r t u r s o n G. P a t h o p h y s io lo g y o f t h e b u r n w o u n d a n d p h a r m a c o lo g i c a l t r e a t m e n t . T h e R u d i H e r m a n s le c t u r e . 1 9 9 5 . B u r n s I 9 9 6 ; 2 2 : 2 5 5 - 2 7 4 . 4 . R u e L W 3 r d , C i o f f i W O J R , R u s h R, M c M a n u s W F, P r u i t t B A J r . T h r o m b o e m b o l i c c o m p lic a t io n s in t h e r m a lly i n j u r e d p a t ie n t s . W o r ld J S u r g 1 9 9 2 ; 1 6 : 1 1 5 1 - 1 1 5 4 . 5 . M u r p y J T , D u f f y 5 , P u r d u e O F, H u n t J L . T h e r m a l i n j u r y a l t e r s e n d o t h e l i a l v a s o c o n s t r i c t o r a n d v a s o d i la t o r r e s p o n s e t o e n d o t o x in . J T r a u m a 1 9 9 9 : 4 7 : 4 9 2 - 4 9 9 . 6 . D r u e c k C, W e lc h G W , P r u i t t B A J r . H e m o d y n a m ic a n a ly s is o f s e p t ic s h o c k in t h e r m a l i n ju r y : t r e a t m e n t w it h d o p a m in e . A m S u r g 1 9 7 8 ; 4 4 : 4 2 4 - 4 2 7 . 7. L a n g C, D o b r e s c u C. A t t e n u a t io n o f b u r n - i n d u c e d c h a n g e s in h e m o d y n a m i c s a n d g lu c o s e m e t a b o l is m b y t h e P A F a n t a g o n is t S R I 6 3 - 6 7 5 . F u r J P h a r m a c o l 1 9 8 8 , 1 5 6 : 2 0 7 . 8 . C i o f f i W G, D e M e u le s J E , G a m e l li R L. V a s c u la r r e a c t i v it y f o l l o w i n g t h e r m a l in ju r y . C ir c S h o c k 1 9 8 8 : 2 5 : 3 0 9 - 3 1 7. 9 . L u k o i a n o v a T l, R o z e l's k a ia L V . F u n c t io n a l d a m a g e o f b l o o d v e s s e ls a n d i n t r a v a s c u la r p l a t e l e t a g g r e g a t io n in b u r n s . B u l l E k s p B io l M e d 1 9 8 3 ; 9 5 : 2 0 - 2 2 . 1 0 . M il e s k i W, G a te s B , S ig m a n A , e t a l. I n h i b i t i o n o f le u k o c y t e a d h e r e n c e in a r a b b i t m o d e l o f m a j o r t h e r m a l in ju r y . J B u m C a r e R e h a b il 1 9 9 3 ; 1 4 : 6 1 0 - 6 1 6 . 1 1 . M c G o w a n J E , B a r n e s M W , F i n l a n d H, B a c t e r e m ia a t B o s t o n C i t y H o s p i t a l : o c c u r r e n c e a n d m o r t a l i t y d u r in g 1 2 s e l e c t e d y e a r s ( 1 9 3 5 - 1 9 7 2 ) w it h s p e c ia l r e f e r e n c e t o h o s p i t a l - a c q u i r e d c a s e s . J I n f e c D is 1 9 7 5 ; 1 3 2 : 3 1 6 - 3 3 5 . 1 2 . H u y t in c k H , O f f e r m a n W, G o r is R J. W h o le b o d y i n f l a m m a t io n in t r a u m a p a t ie n t s . A r c h S u r g 1 9 8 8 ; 1 2 3 : 1 5 1 9 - 1 5 2 4 . 1 3 . B o n e RC. T o w a r d a n e p id e m io lo g y a n d n a t u r a l h i s t o r y o f S IR S ( s y s t e m ic i n f l a m m a t o r y r e s p o n s e s e n d r o m e ) . J A M A 1 9 9 2 : 2 6 8 : 3 4 5 2 - 3 4 5 5 . 1 4 . V is e lli S M , S t a n z ia le S, S h u I ts R, K o v a c s WJ, O ls e n H J . C a s t r a t io n a l t e r s p e r i p h e r a l i m m u n e f u n c t i o n in n o r m a l m a l e m ic e . I m m u n o lo g y 1 9 9 5 ; 8 4 : 3 3 7 - 3 4 2 . 1 5 . W a lk e r S E , B e s c h - W i lli f o r d C L , R e is le r D H . A c c e l e r a t e d d e a t h s f r o m s y s t e m i c l u p u s e r y t h e m a t o s u s in H Z B x H Z W F I m ic e t r e a t e d w it h t e s t e s t e r o n e - b io c k in g d r u g f lu t a m i d e . J L a b C lin M e d 1 9 9 4 : 12 4 : 4 0 1 - 4 0 7 . 1 6 . Ş a n T, M o i n i 11, E m e r k R, B il s e l S. P r o t e c t iv e e f f e c t o f d e f i b r o t i d e o n p e r f u s i o n i n d u c e d e n d o t h e l i a l d a m a g e . T h r o m b R e s 2 0 0 0 ; 9 9 : 3 3 5 - 3 4 1 . 1 7. S h a n J , R e s n ic k L M , Q in - Y u e L, X l- C h e n W, B a r b a g a llo M , P a n g PR. V a s c u la r e f f e c t s o f I 7/J e s t r a d io l in m a l e S p r a q u e - D a w le y r a ts . A m J P h y s i o l ( H e a r t C i r c P h y s i o l 3 5 ) 19 9 4 : 2 6 6 : H 9 6 7 - H 9 7 3 . 1 8 . F a r h a t M Y , L a v ig n e M C , R a m w e ll PW . T h e v a s c u la r p r o t e c t i v e e f f e c t o f e s t r o g e n . F A S E B J 1 9 9 6 ; 1 0 ( A p r i l ) : 6 1 5 - 6 2 4 . 1 9 . H a y a s h i T, F u k u t o J M , lg n a r r o L J , C h a d h u r i G. G e n d e r d i f f e r e n c e s in a t h e r o s c l e r o s i s : p o s s ib le r o l e o f n i t r i c o x id e . J C a r d io v a s c P h a r m a c o l 1 9 9 5 ; 2 6 : 7 9 2 - 8 0 2 . 2 0 . M c G ill H C J r , S t e r n M P . S e x a n d a t h e r o s c le r o s is . A t h e r s c le r R e v 1 9 7 9 ; 4 : 1 5 7 - 2 4 2 . 2 1 . M a d d o x YT, F a lc o n J G , R i d in g e r M . C u n a r d C M , R a m w e ll PW . E n d o t h e liu m - d e p e n d e n t g e n d e r d i f f e r e n c e s in t h e r e s p o n s e o f t h e r a t a o r t a . J P h a r m a c o l E x p T ile r 1 9 8 7 ; 2 4 0 : 3 9 2 - 3 9 5 . 2 2 . Z h a n g A , A l t u r a B T , A l t u r a B M . S e x u a l d i m o r p h i s m o f v a s c u l a r s m o o t h m u s c l e r e s p o n s iv e n e s s Is d e p e n d e n t o n a n io n s a n d e s t r o g e n . S t e r o id s 19 9 1 , - 5 6 : 5 2 4 - 5 2 6 . 2 3 . M a t s u d a R, R u f f A , M o r i n e l l i T A , M a t h u r RS, H a lu s k a P V. T e s t e s t e r o n e in c r e a s e s t h r o m b o x a n e A 2 r e c e p t o r d e n s i t y a n d r e s p o n s iv e n e s s in r a t a o r t a s a n d p l a t e l e t s . A m J P h y s io l ( H e a r t C ir c . P h y s io l, 3 6 ) 1 9 9 4 ; 2 6 7 : H 8 8 7 - H 8 9 3 . 2 4 . Ö z v e r i E, B o z k u r t A , H a k la r G, e t a l. E s t r o g e n s a m e l i o r a t e r e m o t e o r g a n i n f l a m m a t i o n i n d u c e d b y b u r n i n j u r y in r a t s . I n f l a m m R e s 2 0 0 1 ; 5 0 : 5 8 5 - 5 9 1 .
