Ef fects of lig a ture-in duced periodontitis in preg nant Wistar rats
Efeito da doença periodontal induzida por ligadura na prenhez de ratas Wistar
Mariane Ponzio de Azevedo Galvão*
Cassiano Kuchenbecker Rösing**
Maria Beatriz Cardoso Ferreira***
AB STRACT: The aim of this study was to eval u ate the in flu ence of ligature-in duced periodontal dis ease in preg nant rats on their new born’s health pa ram e ters. Twenty-four fe male adult Wistar rats were di vided into two groups: the con trol group (G1) and the group that was sub mit ted to den tal lig a tures around sec ond up per mo lars (G2). Af ter the four week pe riod of de vel op ment of periodontitis, the fe male an i mals were mated with male adult Wistar rats. There were no dif fer ences in the body weight of fe males be tween the two groups dur ing mat ing and preg nancy. No dif fer ences were ob -served among the groups in re la tion to the vi a ble new born in dex. How ever, there were dif fer ences in new born birth weight, ex plained by the di verse size of the lit ters. In this study, lig a ture-in duced periodontal dis ease did not pro mote changes dur ing preg nancy that re sulted in low birth weight in new born Wistar rats.
DESCRIPTORS: Periodontitis; La bor, pre ma ture; In fant, low birth weight; Wistar rats.
RESUMO: O ob je ti vo do pre sen te es tu do foi ava li ar a in fluên cia da pe ri o don ti te in du zi da por li ga du ra em ra tas pre nhes so bre pa râ me tros de sa ú de ge ral de seus fi lho tes. Vin te e qua tro ra tas Wis tar de ida de adul ta fo ram di vi di das em dois gru pos: gru po con tro le (G1) e gru po ex pe ri men tal, que re ce beu li ga du ras ao re dor dos se gun dos mo la res su pe ri o res (G2). Após o pe río do de in du ção de pe ri o don ti te (qua tro se ma nas), as ra tas fo ram co lo ca das para cru za men to com ra -tos Wis tar ma chos, adul -tos. Não hou ve di fe ren ça no peso cor po ral das fê me as du ran te os pe río dos de cru za men to e pre nhez. Tam bém não fo ram ob ser va das di fe ren ças en tre os gru pos quan to à taxa de re cémnas ci dos viá ve is. No en -tan to, hou ve di fe ren ça quan to ao peso dos re cém-nas ci dos, sen do tal di fe ren ça ex pli ca da pela va ri a ção no ta ma nho das ni nha das. No pre sen te es tu do, do en ça pe ri o don tal in du zi da por li ga du ra não pro mo veu mu dan ças du ran te a pre -nhez que re sul tas sem em ba i xo peso ao nas cer dos fi lho tes Wis tar.
DESCRITORES: Periodontite; Trabalho de parto prematuro; Recém-nascido de baixo peso; Ratos Wistar.
IN TRO DUC TION
Re cent re search has shown an as so ci a tion be -tween periodontal dis ease and sys temic disturbances2,13,20,26,27
. Spe cial at ten tion has been paid to the as so ci a tion be tween periodontal dis -ease and preterm low birth weight4,5,6,7,19,20,21
. An ex per i men tal study6
has shown that the in -oc u la tion of Porphyromonas gingivalis in a sub cu -ta ne ous cham ber in fe male ham sters was able to pro mote a sig nif i cant de crease in fe tal weight and sig nif i cant in crease in embryolethality rates. This study also showed the pres ence of in flam ma tory me di a tors pros ta glan din E2 (PGE2) and tu mor ne -cro sis fac tor α (TNFα) at higher lev els in the sub cu -ta ne ous cham ber, af ter the in oc u la tion of
Porphyromonas gingivalis. A sig nif i cant as so ci a tion was found be tween high lev els of these in flam -ma tory me di a tors and low fe tal weight and embryolethality6. Fol low ing these ex per i ments, it
has been shown that the in tra ve nous chal lenge of lipopolysaccharide (LPS) of Porphyromonas gingivalis and Esch e richia coli in ham sters, be fore and af ter mat ing, lead to a sig nif i cant de crease in fe tal weight5. Ham sters fed with su crose rich diet, with or with out in oc u la tion with Porphyromonas gingivalis, de vel oped periodontitis. Af ter perio -dontitis was es tab lished, the an i mals were mated and the re sults showed a re duc tion of 17.4% of fe -tal weight when com pared to con trol animals4.
A case-con trol study20 in hu mans has shown that moth ers with 60% of periodontal sites with at least three mil li me ters of at tach ment loss had a 7.5 times higher chance of hav ing ba bies with low birth weight than con trols. Many stud ies have fo cused on the as so ci a tion be tween periodontal pa thol ogy and preg nancy prob lems in humans7,8. Higher lev els of PGE2 in the crevicular fluid were seen more fre quently in the moth ers of low birth weight newborns19. Re cent in ves ti ga tion showed
* Pro fes sor, Dis ci pli ne of Pe ri o don tics, Gama Fi lho Uni ver sity.
the pres ence of fe tal immunoglobulins against periodontal patho gens, sug gest ing a pos si ble bac -te rial chal lenge in the u-terus21.
Al though ev i dence for the as so ci a tion be tween periodontal dis ease and low birth weight is ac cu -mu lat ing with nu mer ous ex per i men tal and clin i cal re ports, this field still lacks ex per i men tal in ves ti ga tions to elu ci date a causeandef fect re la tion -ship.
The aim of this study was to eval u ate the in flu -ence of in duced periodontal dis ease in preg nant rats in new born’s health pa ram e ters.
MA TE RIALS AND METHODS
An i malsTwenty-four ex per i men tally naïve adult fe male Wistar rats (60 days old) were di vided in two equal ex per i men tal groups. The con trol an i mals (Group 1) re ceived stan dard rat lab o ra tory chow and wa ter. In the Group 2, or the lig a ture-in duced periodontal dis ease group, cot ton lig a tures were placed around the sec ond up per mo lars, and the an i mals re ceived stan dard rat lab o ra tory chow and wa ter.
Eight ex per i men tally naïve adult male Wistar rats (60 days old) were used to mate with the fe -male an i mals. The rats born af ter this match were also stud ied.
In duc tion of periodontal dis ease
To in duce periodontal dis ease, cot ton lig a tures were placed around the sec ond up per mo lars, in rats from Group 2. All rats, even those from Group 1, re ceived gen eral an es the sia with so dium thiopental at the time of lig a ture place ment. A pe riod of one month was es tab lished for the de vel op ment of periodontitis, dur ing this time the pres -ence of lig a tures was checked ev ery week12,24,25,29
. The lig a tures were kept un til the last day of breast feed ing, when the moth ers were sac ri ficed, the maxillae were re moved, fixed, decalcified with 5% ni tric acid, and sec tioned se ri ally at 5 µm in the mesiodistal di rec tion. The sec tions were stained with hematoxylineosin. As part of an other ex per i -men tal re search, a trained ex am iner who was blind to the groups car ried out de scrip tive histological anal y sis in the light mi cro scope. The histological fea tures of 220 histological pieces showed that sam ples from Group 1 (with out lig a -tures) had no signs of in flam ma tion (in tact sulcular ep i the lium with stan dard di men sions; stan dard quan tity of tis sue cells and blood ves sels;
dense supracrestal and periodontal fi bers and reg -u lar al ve o lar bone crests), while sam ples from Group 2 (with lig a tures) showed signs of in flam ma tion (long junctional ep i the lium; over abun -dance of con nec tive tis sue cells and blood ves sels; loose supracrestal and al ve o lar fi bers; ir reg u lar al -ve o lar bone crest with Howship’s la cu nae).
Fre quency of eval u a tion
The body weight of the mother rats was eval u -ated dur ing all the ex per i ment, at a fre quency of three times per week. New borns were checked on the day of birth and three times per week un til they reached the age of 60 days.
Health pa ram e ters
Body weight from moth ers and young rats was mea sured. Right af ter birth, the num ber of young rats per lit ter, the num ber of vi a ble young an i mals per group, and the mean body weight per group were eval u ated. The num ber of vi a ble young an i mals was mea sured as the num ber of vi a ble new -borns di vided by the to tal of new -borns mul ti plied by 100, for each lit ter.
Sta tis ti cal anal y sis
Data were ex pres sed as mean and stan dard de -vi a ti on (SD). Stu dent’s t-test was used to com pa re bet we en two groups. Re pe a ted com pa ri sons were analy zed by re pe a ted me a su res (ANOVA). Li ne ar re gres si on was used to analy ze the in flu en ce of lit -ter size over new born we ight.
RE SULTS
The mot hers’ he alth pa ra me ters are shown in Graphs 1 and 2. No sta tis ti cally sig ni fi cant dif fe -ren ce was ob ser ved in body we ight bet we en the two groups, at the dif fe rent ti mes of eva lu a ti on – du ring pe ri o don tal di se a se in duc ti on, ges ta ti on, af ter de li very, and du ring bre ast fe e ding pe ri ods (one-way ANOVA, p > 0.05).
Ta ble 1 shows the mean and stan dard de vi a ti on of the to tal num ber of ba bi es per lit ter and the vi a ble ba bi es. The re were sig ni fi cant dif fe ren ces bet -we en the groups in re la ti on to the to tal num ber of ba bi es per lit ter (Stu dent’s t-test, p = 0.05), and no dif fe ren ce in re la ti on to vi a ble ba bi es (Stu dent’s
t-test, p > 0.05). The body we ight of ba bi es at birth and af ter bre ast fe e ding is shown in Ta ble 2, whe re we can ob ser ve sta tis ti cally sig ni fi cant dif fe ren ces (Stu dent’s t-test, p < 0.05). The num ber of ba bi es dif fe red bet we en the time of birth and the time af -ter bre ast fe e ding, as some were sa cri fi ced with the
aim of ke e ping the lit ter with the same num ber of new borns1. No sig ni fi cant dif fe ren ce was seen bet we en the groups, in re la ti on to the baby’s body we -ight at 60 days of life (Ta ble 3).
DIS CUS SION
The lit er a ture sug gests a re la tion ship be tween periodontal dis ease and sys temic
pathologies2,16,20,26,27, in clud ing preterm low birth weight problems7,8,19,20,21
.
This study was de signed to un cover pos si ble re -la tions be tween periodontal dis ease in duced in rats and preterm low birth weight. To in duce periodontal dis ease in the fu ture mother rats, cot -ton lig a tures were used around the sec ond up per molars12,24,25,29. The place ment of a lig a ture around the rat’s mo lars pro mote an in flam ma tory chal -lenge with the pres ence of neu tro phils, T- and B-lym pho cytes, ul cer ation and api cal mi gra tion of ep i the lial attachment14. As pre vi ous stud ies have shown im pos si bil ity of pro mot ing periodontitis by place ment of lig a tures in germ-free rats or in rats un der an ti bi otic ther apy, it may be con cluded that lig a ture-in duced periodontal dis ease only oc curs in the pres ence of bacteria24,25
. In con ven tional rats, early plaque is dom i nated by strep to cocci, but as plaque thick ness in creases, Gram-neg a tive
GRAPH 1 Mot her’s body we ight du ring in duc ti on of pe ri o don tal di se a se (mean). Group 1: con trol; Group 2: li -ga tu re.
GRAPH 2 - Mot her’s body we ight du ring the pe ri od of bre ast fe e ding (21 days) (mean). Group 1: con trol; Group 2: li ga tu re.
TABLE 1 - Mean num ber (n) of young rats per lit ter and num ber of vi a ble baby ani mals per lit ter (mean ± SD).
Group n/litter Number of
babiesa
Number of viable babies
(%)b
G1 – control 11 12.00
±
2.05 98.45±
3.50G2 – ligature 11 10.00
±
2.45 98.90±
3.62SD: standard deviation. aSignificant dif fer ence be tween groups (Stu dent’s t-test, p = 0.05). bNo sig nif i cant dif fer ence be tween groups (Stu dent’s t-test, p = 0.76).
TABLE 2 - Body we ight of the baby rats at birth and af ter bre ast fe e ding pe ri od (mean ± SD).
Group n Body weight
at birth (g)a
n (young)
Difference of body weight between birth and 21st
day of life (g)b
G1 – con trol 132 5.57
±
0.52 66 39.50±
5.59G2 – li ga tu re 110 5.90
±
0.49 65 41.67±
4.36SD: standard deviation. aStatistically sig nif i cant dif fer ence (Stu dent’s t-test, p = 0.000; by lin ear lo gis tic re gres sion, con trol ling for lit ter size, p = 0.032). bStatistically sig nif i cant dif fer ence (Stu dent’s t-test, p = 0.02).
TABLE 3 - Body we ight of fe ma le and male baby rats at 60 days of life (mean ± SD).
Group n
(female)
Body weight (g)a
n (male)
Body weight (g)b
G1 –
con trol 34 172.82
±
5.90 32 255.94±
24.98G2 –
li ga tu re 30 179.63
±
26.55 35 261.22±
23.22and an aer o bic or gan isms be come more prom i nent and Bacteroides spe cies are seen in one-month-old rats14.
There was no sig nif i cant dif fer ence be tween groups con cern ing the mother’s weight, in the be -gin ning of the ex per i ment or dur ing periodontal dis ease in duc tion, preg nancy and breast feed ing pe ri ods, which mean that the in duc tion of treat -ments did not bias the re sults.These pa ram e ters are rec og nized as in flu enc ing treat ment and health conditions9,11.
There was no sig nif i cant dif fer ence be tween groups in re la tion to the num ber of vi a ble youngsters28, sug gest ing that the treat ments did not change the mor tal ity num ber at birth. This re -sult is in dis agree ment with a pre vi ous study6, where it was ob served that periodontal patho gens (Porphyromonas gingivalis) in jected in sub cu ta ne ous cham ber in ham sters pro moted embryo -lethality. That study found less new borns per lit ter in ham sters that re ceived Porphyromonas gingivalis, com pared to con trols. An other ex per i -men tal study5 showed that the sublingual ve nous in jec tion of 30 µg of Esch e richia coli lipopoly -saccharide in preg nant ham sters was able to kill the fe tus, with some an i mals show ing uterus dis -charge. The au thors sug gested that oral bac te ria would be able to change the uterus ho meo sta sis. From their re sults, it may be sup posed that bac te rial in jec tion in an i mals may pro mote uterus al ter -ations, which could be the rea son for fe tus death. Uterus and kid ney in fec tions are known to cause preterm low birth weight in hu man beings10,15,17,18,22,23. The dif fer ences found be tween these ex per i men tal stud ies and ours may be re -lated to the dif fer ent an i mal spe cies uti lized, rats
ver sus ham sters. More over, the bac te ria chal lenge was dif fer ent. The sub cu ta ne ous or in tra ve nous in oc u la tion of periodontal patho gens may rep re -sent an acute and spe cific mi cro bial chal lenge, and may re sult in a much more in tense sys temic re sponse, which could al ter the preg nancy out come. In the lig a ture model, periodontal de struc -tion was ob served, but the sys temic re ac -tion was
prob a bly lower. The mean birth weight in the pres -ent study was higher in the group with lig a tures. This find ing is dif fer ent from the lit er a ture. This dif fer ence could re sult from the fact that this group had less new borns per lit ter, with sta tis ti cal sig nif i cance when com pared to the other group. The dif fer ence be tween this find ing and the two ex -per i men tal stud ies shown in the literature5,6 may be re lated to the dif fer ent chal lenge pro mot ing the periodontal dis ease con di tion. The pre vi ous stud -ies were able to ob serve preterm low birth weight when ham sters were in oc u lated with periodontal patho gens, how ever periodontal in flam ma tion caused by cot ton lig a tures was not enough to pro -mote preterm low birth weight in Wistar rats in the pres ent ex per i ment.
Based on the med i cal literature3,10,23, it has been spec u lated that periodontitis in preg nant women may en hance the bac te rial or in flam ma tory me di a -tor chal lenge which can get into the blood stream and cause preterm mem brane rup ture and birth, and as a con se quence low birth weight. Offenbacher et al.19 (1998) found higher lev els of PGE2 and TNF
α
in the amniotic fluid of an i mals with periodontitis.In con trast to what the lit er a ture speculates4,5,6, it was ob served in the pres ent study that periodontal dis ease in duced by lig a ture pro moted no al ter ations in preg nant rats, and nei ther caused preterm low birth weight. There was no ef -fect of the treat ment – lig a ture – upon the body weight of mother rats dur ing the dif fer ent ex per i -men tal pe ri ods. The num ber of new borns was higher in the con trol group. But the num ber of vi a -ble new borns was sim i lar be tween the dif fer ent groups. At birth and af ter breast feed ing lag, the new borns from moth ers sub mit ted to periodontal in duc tion with lig a ture had the high est mean birth weight, but this was in flu enced by lit ter size.
CON CLU SION
The re la tion of cause and ef fect be tween periodontitis and preterm low birth weight has not been shown in the pres ent study, ac cord ing to the meth od ol ogy of this ex per i ment.
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