Morphological Changes and Vascular Reactivity of Rat Thoracic Aorta Twelve Months After Pinealectomy

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1 ORİJİNAL ARAŞTIRMA Morphological Changes and Vascular Reactivity of Rat Thoracic Aorta Twelve Months After Pinealectomy Zehra KURÇER, MD, a Feral ÖZTÜRK, MD, b Engin ŞAHNA, MD, c Meltem KURUŞ, MD, b Ercüment ÖLMEZ, MD d a Department of Pharmacology, Zonguldak Karaelmas University Faculty of Medicine, Zonguldak b Department of Histology and Embryology, İnönü University Faculty of Medicine, Malatya c Department of Pharmacology, Fırat University Faculty of Medicine, Elazığ d Department of Pharmacology, Celal Bayar University Faculty of Medicine, Manisa Ge liş Ta ri hi/re ce i ved: Ka bul Ta ri hi/ac cep ted: Ya zış ma Ad re si/cor res pon den ce: Zehra KURÇER, MD Zonguldak Karaelmas University Faculty of Medicine, Department of Pharmacology, Zonguldak, TÜRKİYE/TURKEY zykurcer@yahoo.com ABS TRACT Objective: Melatonin, a hormone produced by the pineal gland, has been suggested to protect against development of hypertension and atherosclerosis. In this study, the effects of longterm melatonin deficiency for twelve months after pinealectomy on the α-adrenergic-contractions induced by phenylephrine, endothelium-dependent relaxation responses to acetylcholine and the morphological changes in the rat thoracic aorta were studied. Material and Metods: Rats were pinealectomized twelve months before the beginning of the vasomotor studies. Rings of arteries were mounted in isolated tissue baths for the measurements of isometric contractile force. The contractile responses to phenylephrine and endothelium-dependent relaxation responses to acetylcholine in the vessels were evaluated. Endothelial function was evaluated by vascular relaxation to acetylcholine. Histological examinations demonstrated the alterations of tunica media in the vessels of pinealectomized rats. Results: Thick and thin areas were observed in the transverse sections of vessels and the ratio of the widest media thickness to the narrowest was found significantly increased in pinealectomized group (2.85 ± 0.56) when compared to the control group (1.65 ± 0.10). In addition, α-smooth muscle actin and elastic lamellae staining of the media were attenuated in pinealectomized rats. Although contractile responses of vessels to phenylephrine in pinealectomized rats were lower than control group, significant difference was found for only one concentration (3x 10-8 mol l-1) of phenylephrine. There was no difference between the relaxation responses to acetylcholine in pinealectomized and control groups. Conclusion: These results show that long-term melatonin deficiency may cause some morphological changes in the tunica media of vessels. However, the function of endothelium and vascular responsiveness to -adrenergic stimulus seem to be mostly protected. Key Words: Melatonin; aorta, thoracic; rats ÖZET Amaç: Pineal bezden üretilen bir hormone olan melatoninin hipertansiyon ve ateroskleroza karşı koruyucu olduğu ileri sürülmüştür. Bu çalışmada sıçan torasik aortalarında, pinealektomi sonrası on iki ay süreyle uzun süreli melatonin eksikliğinin, fenilefrinle oluşturulan α-adrenerjik kasılmalar, asetilkolinle oluşan endotelyuma bağlı gevşemeler ve morfolojik değişiklikler üzerine etkileri çalışıldı. Gereç ve Yöntemler: Sıçanların pineal bezleri vazomotor çalışmaların başlamasından on iki ay önce çıkarıldı. Arter halkaları izometrik kasılma gücün ölçümü için izole doku banyosuna asıldı. Damarlarda fenilefrinle oluşturulan kasılma cevapları ve asetilkolinle oluşan endotelyuma bağlı gevşeme cevapları değerlendirildi. Pinealektomize sıçanların damarlarındaki tunika media tabakasının değişiklikleri histolojik olarak muayene edildi. Bulgular: Kontrol grubu (1.65 ± 0.10) ile karşılaştırıldığında damarların transvers kesitlerinden elde edilen kalın ve ince alanlar ve media tabakasının en geniş kalınlığının en dara oranı pinealektomize grupta (2.85 ± 0.56) anlamlı olarak artmış bulundu. Ayrıca, medianın α-düz kas aktin ve elastik lamel boyaması pinealektomize sıçanlarda azaldı. Her ne kadar pinealektomize sıçanlardaki arterlerin fenilefrine kasılma cevapları kontrol grubundan daha düşük olsa da fenilefrinin yanlızca bir konsantrasyonunda (3x 10-8 mol l- 1) anlamlı farklılık bulundu. Pinealektomize ve kontrol grupların asetilkoline gevşeme cevapları arasında anlamlı farklılık yoktu. Sonuç: Bu sonuçlar uzun süreli melatonin eksikliğinin damarların tunika mediasında bazı morfolojik değişikliklere neden olabileceğini göstermektedir. Bununla birlikte endotelyumun fonksiyonu ve -adrenerjik uyarılara damarsal cevap verirlilik çoğunlukla korunmuş gibi görünmektedir. Anah tar Ke li me ler: Melatonin; aorta, torasik; sıçanlar Cop yright 2010 by Tür ki ye Kli nik le ri :

2 Medical Pharmacology e la to nin, a hor mo ne pro du ced by the pi - ne al gland, is re le a sed with a cir ca di an rhythm, with high le vels at night. 1 Me lato nin has be en sug ges ted as a po tent an ti o xi dant that may pro tect the de ve lop ment of hyper ten si on and at he rosc le ro sis. Dec re a sed me la to nin synthe - sis has be en de ter mi ned in pa ti ents with hyper ten - si on or co ro nary ar tery di se a se. 2,3 Me la to nin ad mi nis tra ti on has be en ob ser ved to re du ce blo od pres su re and dec re a se ca tec ho la mi ne le vels in hu - man sub jects. 4,5 It has al so be en re por ted that me - la to nin re ver sed N-methyl-l-ar gi ni ne in du ced blo od pres su re (BP) ele va ti on, and in cre a sed ca ro - tid ar tery blo od flow in rats. 6,7 Im pa i red en dot he li - al func ti on which may play a ro le in the pat hoph ysi o logy of hyper ten si on 8 and at he rosc le - ro sis, 9 was shown to be res to red by me la to nin pro - bably du e to its an ti o xi da ti ve pro per ti es. 10,11 Mo re o ver, age-re la ted chan ges in the amp li tu de of the me la to nin rhythm in hu mans of ad van ced age ha ve be en re por ted. 12 Thus, it co uld be sug ges ted that dec re a sed me la to nin se rum con cen tra ti ons with aging may re sult in en dot he li al dysfunc ti on and pro mo te hyper ten si on or at he rosc le ro sis. On the ot her hand, an in te res ting in vol ve ment of me la to nin in the car di o vas cu lar re gu la ti on of rats was sug ges ted by pi ne a lec tomy ex pe ri ments that re sul ted in a tem po rary hyper ten si on. 13 In cre - a sed BP le vels ha ve be en re por ted to re turn to the nor mal ran ge two months af ter pi ne a lec tomy in rats Cun na ne et al. re por ted that va so cons tric - tor res pon ses to no re pi nep hri ne, se ro to nin and angi o ten sin II in pi ne a lec to mi zed (Px) rats we re gre a ter than that of con trol rats a we ek af ter pi ne - a lec tomy, i.e. in the short-term pe ri od of pi ne a lec - tomy. 17 Za no bo ni et al. al so sho wed the wall thic ke ning and lu men nar ro wing of the rat ar te ri - o les 90 days af ter pi ne a lec tomy. 13 Ho we ver, the re is no da ta abo ut the ef fects of dec re a sed me la to nin le - vels on the en dot he li al func ti on, vas cu lar re ac ti vity and morp ho logy of ves sels in a lon ger pe ri od after pi ne a lec tomy. The pre sent study was de sig ned to in ves ti ga te the ef fects of re du ced me la to nin le vels twel ve months af ter pi ne a lec tomy, which is a qu i te long pe ri od for rat stu di es, on the α-ad re ner gic-in du ced Kurçer et al con trac ti ons, en dot he li um-de pen dent re la xa ti ons and morp ho lo gi cal chan ges in the rat tho ra cic aor - ta. MA TE RI AL AND MET HODS ANI MALS Twenty ma le Wis tar rats we ig hing g we re pla ced in a qu i et and tem pe ra tu re (21 ± 2 C) and hu mi dity (60 ± 5%) con trol led ro om in which a h light-dark cycle was ma in ta i ned. All ex pe ri ments in this study we re per for med in ac cor dan ce with the gu i de li nes for ani mal re se - arch from the Na ti o nal Ins ti tu tes of He alth and we - re ap pro ved by the Com mi te e on Ani mal Re se arch at Ino nu Uni ver sity, Ma lat ya. PI NE A LEC TOMY Rats we re pi ne a lec to mi zed twel ve months be fo re the be gin ning of the va so mo tor stu di es and morp - ho lo gic eva lu a ti on of their aor tas, in or der to cons ti tu te a long term me la to nin de fi ci ency sin ce the amp li tu de of the me la to nin rhythm has be en shown to be re du ced by % af ter pi ne a lec - tomy in pre vi o us stu di es. 1 Pi ne a lec tomy was per for med for ten rats as des cri bed by Kus zak and Ro din. 18 Rats we re anest he - ti zed with ke ta mi ne hydroc lo ri de (75 mg kg -1 ) and xyla zi ne (8 mg kg -1 ) be fo re the ope ra ti on. The enti re pro ce du re was comp le ted wit hin 15 min. Pi - ne a lec tomy was con fir med by the his to lo gi cal eva lu a ti on of the gland for each ani mal. Ten rats, as a con trol gro up, un der went sham ope ra ti ons. VA SO MO TOR STU DI ES Freshly har ves ted tho ra cic aor tas of rats we re cle - a ned of fat and con nec ti ve tis su es. Two rings (each ap prox. 0.5 cm long) we re pre pa red from a seg ment of tho ra cic aor ta. Pre pa ra ti ons we re mo un ted in 40-ml or gan baths con ta i ning Krebs-Hen se le it buf - fer (mmol l -1 : NaCl 118, KCl 4.7, CaCl 2 2.5, MgSO 4 1.2, KH 2 PO 4 1.2, NaH CO 3 25, glu co se 11, ph 7.4) ma in ta i ned at 37 C and oxy ge na ted with 95% O 2, CO 2 mix tu re. The pre pa ra ti ons we re sus pen ded un - der 0.5 g res ting ten si on which was de ter mi ned in the ba se li ne stu di es and equ i lib ra ted for 60 min, 617

3 Kurçer ve ark. with replacement of bat hing flu id every 15 min. Iso met ric ten si on stu di es we re per for med using a Har vard Uni ver sal mo del os cil log raph (Har vard Ap pa ra tus, Inc., Mas sac hu setts, U.S.A). Cu mu la ti ve con cen tra ti on-res pon se cur ves to pheny lep hri ne (Phe, 10-8 to 10-4 mol l -1 ) we re estab lis hed. The ves sels we re then sub ma xi mally precon trac ted with Phe (typi cally mol l -1 ), and en dot he li al func ti on was eva lu a ted by vas cu lar rela xa ti on to acetyl cho li ne (Ach, 10-8 to 10-4 mol l -1 ). Nit ric oxi de (NO) me di a ti on of ACh res pon ses was con fir med by bloc king ACh-in du ced re la xa ti ons by N-methyl-l-ar gi ni ne (L-NA ME, 10-3 mol l -1 ), a speci fic com pe ti ti ve in hi bi tor of NO syntha se. Con trac ti le res pon ses we re me a su red as for ce (g) and ex pres sed as a per cen ta ge of the ma xi mal con trac ti on or, for re la xa ti ons, as a per cen ta ge of the pre con trac ted ten si on. HIS TO LOGY For light mic ros co pic eva lu a ti on, aor ta sec ti ons we - re fi xed in 10% phosp ha te buf fe red for ma lin. Pa - raf fin-em bed ded spe ci mens we re cut in to 5mm thick sec ti ons and sta i ned with He ma toxy li n e o - sin (H-E), Mas son s tric hro me sta in, Ver ho eff s elas tic sta in and an ti a-smo oth musc le ac tin im mu - nos ta i ning kit (Sig ma Chem. Co. St. Lo u is). The aor ta sec ti ons we re exa mi ned with Le i ca DFC 280 light mic ros co pe by an ex pe ri en ced ob ser ver una - wa re of the ani mal tre at ment gro ups. Le i ca Q Win Plus Ima ge Analy sis System (Le i ca Mic ros Ima ging So lu ti ons Ltd.; Cam brid ge, U.K.) was used for morp ho met ric analy sis. Ten dif fe rent sec ti ons we re me a su red (µm) for each rat. The me a su re ments we - re app li ed to tu ni ca me di a in 10 dif fe rent fi elds for each sec ti on. The ra ti os of the mi ni mum and ma x- i mum va lu es for each sec ti on we re se lec ted for statis ti cal analy sis. Tıbbi Farmakoloji ex pe ri ments. Dis tri bu ti on of the samp les in the gro ups was analy zed with one samp le of Kol mo go - rov-smir nov test. Sta tis ti cal analy sis of the da ta was per for med by using Stu dent s t test for va so mo tor fin dings and by Mann-Whit ney U test for his to lo - gi cal fin dings. The dif fe ren ces bet we en gro ups of da ta we re con si de red to be sig ni fi cant when p< RE SULTS The re was no dif fe ren ce bet we en the body we ights of Px (167 ± 6.9 g) and con trol (173 ± 7.5 g) gro ups be fo re the pi ne a lec tomy or sham ope ra ti on. Alt ho - ugh body we ights of Px rats se e med to be in cre a sed, no sig ni fi cant dif fe ren ce was de mons tra ted bet we - en Px (414 ± 15 g) and con trol (398 ± 12 g) ani mals twel ve months af ter the ope ra ti on. Phe pro du ced con cen tra ti on-de pen dent contrac ti ons in tho ra cic aor tas iso la ted from con trol and Px rats. Alt ho ugh con trac ti le res pon ses of vessels to Phe in Px rats we re lo wer than con trol gro - up, sta tis ti cally sig ni fi cant dif fe ren ce was fo und for only one con cen tra ti on (3x 10-8 mol l -1 ) of Phe (Fi- gu re 1). On the ot her hand, ma xi mum con trac ti le res pon ses to Phe in the tho ra cic aor tas of Px rats (0.91 ± 0.11 g) we re not sig ni fi cantly dif fe rent from the con trol rats (0.93 ± 0.07 g). En dot he li um-de pen dent re la xa ti on res pon ses to Ach al so ten ded to dec re a se in Px rats, whi le no sig ni fi cant dif fe ren ce was de ter mi ned bet we en two DRUGS Phe, ACh and L-NA ME we re purc ha sed from Sig - ma Che mi cal Co. (St. Lo u is, MO, USA). All drugs we re dis sol ved in dis til led wa ter. Statistical Analysis Re sults we re ex pres sed as the arith me tic me an ± standart error of the mean (SEM) of the num ber of FIGURE 1: Concentration-response curves for phenylephrine (Phe)-induced contractions in isolated aorta rings of control (open circles) and pinealectomized (solid circles) rats. Each point represents the mean ± SEM bars of 10 experiments. *Significantly different from the values of control group. 618

4 Medical Pharmacology FIGURE 2: Concentration-response curves for endothelium-dependent relaxations induced by acetylcholine (ACh) in isolated aorta rings of control (open circles) and pinealectomized (solid circles) rats. All vessels were submaximally precontracted by 3x10-7 mol l-1 phenylephrine (Phe). Each point represents the mean ± SEM bars of 10 experiments. gro ups (Fi gu re 2). Re la xa ti ons to Ach we re comp - le tely in hi bi ted by L-NA ME, thus con fir ming the - ir de pen den ce on NO pro duc ti on (da ta not shown). Tu ni ca in ti ma, me di a and ad ven ti ti a of all rat tho ra cic aor ta spe ci mens sho wed nor mal his to logy in the con trol gro up (Fi gu re 3a). In the Px gro up, the in ti ma and ad ven ti ti a we re al so nor mal. No evi den ce of dis rup ti on and at he rosc le ro sis was de tec - ted in the in ti ma (Fi gu re 3b). Ho we ver, the thick ness of the me di a in Px rats sho wed al te ra ti - ons. In the trans ver se sec ti ons, thick ness of the vessel wall was not the sa me along its di a me ter; thick and thin are as we re ob ser ved (Fi gu re 4a). The ra ti - o of the wi dest me di a thick ness to the nar ro west was as ses sed and fo und sig ni fi cantly in cre a sed in Px gro up (2.85 ± 0.56) com pa red to that in con trol gro up (1.65 ± 0.10). The aor tas of two rats in Px gro up sho wed pro mi nent wall thin ning (Fi gu re 4b). Although the elas tic la mel la e of con trol gro up we re sta i ned pro mi nently by Ver ho eff s elas tic sta - in, this sta i ning was we ak in the ves sels of Px rats (Fi gu re 5). The an ti α-smo oth musc le ac tin sta i ning was strongly po si ti ve (+++) in the con trol gro up, whe re as the Px gro up sho wed we ak po si ti ve (+) sta - i ning (Fi gu re 6). DIS CUS SI ON In the pre sent study, his to lo gi cal exa mi na ti on re ve - a led the tu ni ca me di a chan ges in the rat tho ra cic Kurçer et al aor ta du e to long-term me la to nin de fi ci ency. In the trans ver se sec ti ons of the ves sels, twel ve months af ter pi ne a lec tomy, thin and thick me di a sec ti ons we re ob ser ved and the ra ti o of the me di a thick ness, i.e. the ra ti o of the wi dest to the nar ro west me a su - re ment, was sig ni fi cantly in cre a sed com pa red to that of con trol rats. Reg rigny et al. re por ted re du - ced wall thick ness in the ce reb ral ar te ri o les of rats one month af ter pi ne a lec tomy. 19 They al so ob ser - ved that tre at ment with me la to nin res to red nor mal wall thick ness. On the ot her hand, Za no bo ni et al. ob ser ved ar te ri o lar wall thic ke ning and lu men narro wing in the kid neys of Px rats 90 days af ter pi ne - a lec tomy. 13 Sin ce the tho ra cic aor ta is an elas tic ar te ri a and its me di a his to logy is dif fe rent from or - gan ar te ri es, this study de mons tra ted for the first ti me the ef fects of long-term pi ne a lec tomy on the morp ho logy of elas tic ar te ri es. We de ter mi ned his to lo gi cal al te ra ti ons in the elas tic la mel la e and smo oth musc le cells of rat thora cic aor ta af ter pi ne a lec tomy. Elas tic la mel la e and smo oth musc le cells are the ba sic com po nents of the tu ni ca me di a in elas tic ar te ri es. Vas cu lar smo - oth musc le cells are ca pab le of many func ti ons inc lu ding synthe sis of col la gen, elas tin and pro te o- gl ycans. They al so cons ti tu te an im por tant ele ment in nor mal vas cu lar re pa ir and in pat ho lo gic pro ces - ses such as at he rosc le ro sis. 20 In the Px gro up, we ob ser ved that smo oth musc le cells we akly sta i ned with an ti α-smo oth musc le ac tin. Elas tic la mel la e of the me di a we re al so thin and we akly sta i ned with elas tic sta in. The se al te ra ti ons might be occur red as a re sult of the smo oth musc le cell da ma - ge as well as im pa ir ment in the func ti on of the se cells. Anot her fin ding of the cur rent study was that the res pon si ve ness to Phe, an µ 1 -se lec ti ve ad re - ner gic ago nist, was at te nu a ted in the aor tic rings of Px rats as com pa red to con trols, alt ho ugh sta tis ti - cally sig ni fi cant dif fe ren ce was fo und for only one con cen tra ti on of Phe. This re sult is dif fe rent from the fin ding that de mons tra ted an en han ce ment of µ 1 -ad re ner gic-in du ced con trac ti ons in rat ves sels one we ek af ter pi ne a lec tomy. 18 In our pre vi o us study, we ob ser ved that re du ced me la to nin le vels in the se cond month fol lo wing pi ne a lec tomy did 619

5 Kurçer ve ark. Tıbbi Farmakoloji FIGURE 3: Photomicrographs of thoracic aorta in control (a) and pinealectomy (b) groups. a: Tunica intima, media and adventitia appear normal. Hematoxylineosin X132. b: Tunica intima is normal. No evidence of atherosclerosis and disruption are seen. The media alteration is not detectable in this figure. Hematoxylineosin X132. FIGURE 4: Photomicrographs of thoracic aorta in pinealectomy group. a: Thick (k) and thin (n) tunica media are seen. Masson s trichrome stain X33. b: Prominent reduction in media thickness is visible. Anti α-smooth muscle actin immunostaining X66. FIGURE 5: Photomicrographs of thoracic aorta in control (a) and pinealectomy (b) groups. a: Thick and continuous elastic lamellae are stained prominently. Verhoeff s elastic stain X66. b: Thin elastic lamellae are stained weakly. Verhoeff s elastic stain X

6 Medical Pharmacology Kurçer et al FIGURE 6: Photomicrographs of thoracic aorta in control (a) and pinealectomy (b) groups. a: The smooth muscle cells are stained strongly. Anti α-smooth muscle actin immunostaining X66. b: The smooth muscle cells are stained weakly. Anti α-smooth muscle actin immunostaining X66. not mo dify the vas cu lar re ac ti vity to va ri o us va so - cons tric tor agents such as pheny lep hri ne, 5-HT, clo ni di ne, AT-II and va sop res sin. 16 As the α-smo - oth musc le ac tin is the ma jor ac tin iso form of vascu lar tis su e and con tri bu tes to cell-ge ne ra ted mec ha ni cal ten si on, 21 dec re a sed α-smo oth musc le ac tin sta i ning of rat aor ta ob ser ved in the pre sent study may exp la in the re du ced res pon ses to Phe fol lo wing long-term me la to nin de fi ci ency. We al so de mons tra ted dec re a sed elas tin compo nent of tho ra cic aor ta in Px rats. Ele va ted ar te ri - al stiff ness was shown to be re la ted to ex tra cel lu ler mat rix elas tin/col la gen ra ti o and ac cep ted as an impor tant pre dic tor of car di o vas cu lar events. Drugs which re du ce the ar te ri al stiff ness ha ve be en shown to in cre a se the elas to ge nic mat rix pro fi le in aor tic smo oth musc le cell cul tu re. 22 On the ot her hand, we hypot he si zed that long-term me la to nin de fi ci ency wo uld ca u se an en dot he li al dysfunc ti on, sin ce the oxi da ti ve stress over the an ti o xi dant ca pa city of the body is ac cep - ted to le ad to en dot he li al dysfunc ti on, 9-11 and to tal an ti o xi dant ca pa city of rat se rum was shown to be re la ted to the se rum con cen tra ti ons of me la to nin. 23 Ho we ver, this pos si bi lity is not li kely be ca u se we de mons tra ted comp le tely in tact vas cu lar en dot he - li um his to lo gi cally twel ve months af ter pi ne a lec - tomy, and we co uld not show any sig ni fi cant re duc ti on in the en dot he li um-de pen dent re la xa ti - on res pon ses to Ach. In conc lu si on, the pre sent study de mons tra ted that long-term me la to nin de fi ci ency for twel ve months fol lo wing pi ne a lec tomy ca u sed so me signi fi cant chan ges in the tu ni ca me di a of the rat thora cic aor ta such as al te red me di a thick ness and dec re a sed α-smo oth musc le ac tin and elas tic la mella e sta i ning. On the ot her hand, the in teg rity and func ti on of en dot he li um and vas cu lar res pon si ve - ness to µ-ad re ner gic sti mu lus se em to be mostly pro tec ted. The im por tan ce of the morp ho lo gi cal chan ges in the tu ni ca me di a of ves sels is not cle ar. Ack now led ge ments This study was sup por ted by a grant from The Sci en ti fic Re se arch Fund of Ino nu Uni ver sity (P-01/33). We thank Prof. Dr. Sa im Yo log lu for sta tis ti cal analy sis. 621

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