periodontal disease is a multifactorial infectious disease triggered by the development of dental biofilm , which can harm periodontal tissues . there are several factors which influence periodontitis : personal and social characteristics , behavioral , systemic , genetic and dental factors , and the microbial composition of the dental biofilm [ 1 , 2 ] . stress may be defined as the combination of physical and mental responses caused by certain external stimuli ( stressors ) which allow an individual ( human or animal ) to overcome certain environmental demands , and the physical and mental weariness caused by the stress process itself . stress has been related not only to a higher occurrence of periodontal disease but also to its severity . other social and behavioral factors that could be related to periodontal status are tobacco use , social economic status , nutritional status , psychological aspects , and abusive alcohol intake . among these factors , alcohol has become relevant because it is one of the few socially accepted psychotropic drugs . this social acceptance has contributed to a rise in alcoholism , which has become a serious public health problem . according to silva furtado amaral et al . the world health organization has reported that the rates of death and functional limitations associated with abusive alcohol intake have surpassed those associated with tobacco smoking . alcoholism per se imposes a state of stress because , besides its physiological demand , it acts as a chemical aggressor to the body . all the alcoholic drinks consumed by an individual do not remain stored in the body and , before being eliminated , it is first metabolized in the liver , which results in high levels of reactive oxygen species [ 7 , 8 ] . several cross - sectional studies have verified an association between the abusive consumption of alcohol and poor periodontal health [ 911 ] . abusive consumption of alcohol has also been associated with the severity of periodontitis . however , many questions on this subject are still not answered and laboratorial research could be helpful to clarify these doubts . animal model studies may more specifically help elucidate questions related to alcohol intake , stress , and periodontal disease [ 12 , 13 ] . as a lifestyle factor drinking , and stress seem to contribute to poorer periodontal status . in the present study it was hypothesized that alcohol consumption by stressed animals could be accompanied by an increase in the severity of periodontitis . therefore , aiming to confirm or reject this hypothesis we evaluated whether forced alcohol intake by stressed animals affects histometrical parameters of periodontal breakdown . thirty - two animals of the rattus norvegicus species lewis breed ( two - month old ) , weighing average 250  g , were selected for the present study . the animals were divided in groups of four and each group was kept in a cage ( polyethylene 16  40  30 ) [ 14 , 15 ] . they all received standard rat feed and water or alcohol ( ethanol 20% ) ad libitum . they were kept in light / dark cycles of 12 hours ; the temperature was controlled at 24c and humidity at > 50% . the experiment took place at the university center of vrzea grande  univag and it was approved and registered by the ethics in animal research committee ( cep / unic-2009 no . initially , a research assistant randomly divided the animals in four experimental groups : group gal : alcohol + ligature ( n = 8);group gasl : alcohol + stress + ligature ( n = 8);group gpc : stress + ligature , positive control ( n = 8);group gcn : negative control ( n = 8) . group gal : alcohol + ligature ( n = 8) ; group gasl : alcohol + stress + ligature ( n = 8) ; group gpc : stress + ligature , positive control ( n = 8) ; group gcn : negative control ( n = 8) . after the division , the animals in the gal and gasl groups were offered alcohol ( ethanol 20% ) ad libitum for 60 days [ 11 , 16 ] while negative control ( gcn ) received water ad libitum for the same period of time . because alcohol intake is very limited on the first day , ligatures were inserted only in the second experimental day ( gal , gasl , and gpc ) . the animals received general anesthesia through the intramuscular administration of 0.05  ml xylazine hydrochloride ( rompun , bayer . animal health , so paulo , sp . brazil ) and 0.1  ml ketamine ( dopalen , agribrands . animal health , paulnia , sp , brazil ) per 100 grams of body weight . after anesthesia , a sterile silk suture number 4 ( ethicon , johnson and johnson , so paulo , sp , brazil ) was placed in the gingival crevice of the second right upper molar . the animals in the positive control group were kept in the same environment in the first experimental day , drinking water , but without suffering any kind of intervention . the animals in the negative control group ( gnc ) did not receive any type of intervention but were kept in their cages in the same environment during all the study . the model of physical stress induction chosen for this study was immobilization done during 60 days , six times a week , in different times of the day as previously described [ 18 , 19 ] . at room temperature , the animals in the gasl group were placed in polyvinyl chloride tubes compatible to their body sizes . the tubes were closed on both ends with metallic wire , which enabled the animals to breathe while they were immobilized for four consecutive hours . after that process , the jaw segment was decalcified in edta for approximately five weeks ( edta was renewed six times ) ; following dehydration in graded alcohol , the samples were embedded in paraffin . the tissue blocks included in paraffin allowed 6  m histological slices that followed the long axis of the teeth in the mesio - distal plane and were stained with hematoxylin and eosin .   for the histometric analysis , ten serial sections containing the 1st and 2nd upper molars and the following structures ( a ) dental pulp , ( b ) the mesial cementoenamel junction of the 2nd molar , ( c ) proximal bone crest , and ( d ) connective attachment had to be observed . histometry was performed in the mesial aspect from first molars , by the same blinded examiner , through the capturing of images in the microscope and measured in millimeters ( software imagelab 2000-diracon bio informtica ltda . previous to the study , the error was calculated by double measurements of 10% of the specimens with a one - week interval . during the study , paired t - test statistics were run and no differences were observed in the mean values for comparison ( p > 0.5 ) . additionally , pearson 's correlation coefficient was obtained between the 2 measurements in each time point and revealed a very high correlation ( 0.98 and 0.99 ; p < 0.01 ) . the distance between the cementoenamel junction and the bottom of the junctional epithelium ( cej - je ) in the mesial site of the first molars was defined as the loss of histological attachment . in addition , the distance between the cementoenamel junction and the alveolar bone crest was also analyzed and it was considered as the indicator for bone loss ( cej - bc ) . analysis of variance ( anova ) and tukey ( p < 0.05 ) tests were applied . there were no statistically significant differences among the body weights of the groups at any of the evaluation periods ( anova , p < 0.05 ) . loss of histological attachment ( cej - je ) and bone loss ( cej - bc ) ( p < 0.05 ) were statistically different in the animals exposed to alcohol ( gal ) and the ones exposed to alcohol and stress ( gasl ) . the most severe periodontal breakdown was observed among the nonstressed animals exposed to alcohol , followed by the group of stressed animals exposed to alcohol . this last group ( gasl ) did not differ from the positive control group ( gpc ) . figures 1 and 2 illustrate results from gasl group at two different experimental times . finally , the negative control group showed the lowest values of cej - je and cej - bc when compared to the animals in the other groups ( table 1 ) . the severity and rate of the progression of periodontitis are influenced by a great variety of determinants and risk factors . oral hygiene , tobacco smoking , diabetes , age , and the presence of specific microorganisms can be considered as risk factors , since individuals under these conditions seem to be more likely to develop periodontitis . however , the combination of these factors is not enough to explain the difference in the progression rate and severity of the disease . other systemic conditions such as stress , osteoporosis , obesity , and abusive alcohol drinking   have been considered as partially responsible for this variability as well . the effects of alcohol to the body and the functional imbalances associated with its consume have been constantly investigated . such interest is justified by the wide range of injuries and toxic effects that alcohol drinking causes the organs and tissues , which can reflect in different systemic diseases . besides , the world health organization estimates that two billion people around the world are alcohol consumers , and that 76 million show some kind of systemic adverse event related to that . in brazil , it is estimated that 68% of the population consumes alcohol and that 11.2% are alcoholics . de souza et al .   demonstrated that alcohol consumption directly framed the progression of bone loss in an induced periodontitis model . on the other hand , instead of a direct action of alcohol over the periodontal tissues , these effects are based on poor oral hygiene , which could better explain a higher occurrence and severity of periodontal disease in people who drink alcohol . in this context , animal studies are important as they may help understand the biological events once they allow the elimination of certain bias by the control of some variables . therefore , animal studies have observed that alcohol affected the periodontal tissues through different mechanisms . reduced host responses , due to deficiency and functional changes in neutrophils , as well as changes in the protein metabolism and in the healing of tissues have been previously reported . in the present study alcohol drinking negatively impacted the progression of periodontitis . similarly de souza et al .   also observed a direct effect of alcohol drinking in the bone loss progression in a ligature - induced periodontitis model . one common question is in which level the alcohol as a water substitute under experimental conditions reflects human alcohol drinking . the answer to that is not simple , especially considering that the metabolism of rats is faster than that of humans . according to the world health organization mild drinkers are men who drink 21 alcohol units ( 1 unit = 10 grams ) per week . for women , for example , a glass of red wine ( 90  ml ) has 12% or 1.7 unit of alcohol . a beer has 5% ( 1.1 unit ) while distilled drinks such as vodka and whisky have 40% or 2.0 alcohol units . supporting dose dependence , de souza et al . reported a significantly higher alveolar bone loss in rats receiving 20% ethanol in comparison to rats receiving 10% ethanol or water . .   carried an interesting cross - sectional study to evaluate the effect of the consumption of different alcoholic drinks ( wine , beer , and liquor ) on the severity of periodontal disease . they observed that the effects over the periodontal tissues did not change depending on the kind of drink taken . these same authors also verified contrary activity of alcohol over some periodontal pathogens such as actinobacillus actinomycetemcomitans and porphyromonas gingivalis . clinical results suggested that alcohol seems to affect more severely the gums , followed by the periodontal ligament and the alveolar bone and that the effect of alcohol on periodontal disease may depend on the dose , frequency , and time of drinking . .   also demonstrated that individuals that consumed more than 15 grams of alcohol a day had a significant increase in the progression rate of periodontal disease and a higher inflammatory infiltrate , besides a higher number of periodontal pockets when compared to those who did not consume alcohol . although , dental biofilm acts besides some recognized risk factors such as tobacco use , as previously mentioned , more recently , researchers are considering whether and in what degree lifestyle could affect the periodontium . in this context , stress seems to affect not only periodontal status but also personal lifestyle . individuals exposed to stressful situations tended to smoke a higher number of cigarettes   and become alcohol consumers . also , a decrease in oral hygiene was observed in a group of stressed individuals . furthermore , direct tissue damage could be attributed to the stress process . due to the frequent combined occurrence of the studied factors , this study evaluated the effect of alcohol intake by stressed animals . the fact that the stressed animals exposed to alcohol showed better periodontal conditions than the nonstressed animals was only a partially unexpected result of this study . yaroslavsky and tejani - butt   evaluated the relation between stress and alcohol consumption and observed that rats exposed to chronic stress consumed a greater amount of alcohol . in addition , changes in central dopamine type-2 receptor sites were found indicating an altered dopamine neurotransmission following stress and alcohol exposure . since stressed animals consumed more alcohol , the authors concluded that it is possible that the consumption of alcohol reverses these alterations related to dopamine , suggesting a self - medicating phenotype . compared the immunological effect of the discontinuous feeding of a liquid diet containing a moderate amount of ethanol to that of continuous ethanol administration or a control diet . the discontinuous alcohol group received the ethanol diet for 3 days and the control liquid diet for the remaining 4 days of each week ( for a total of 4 weeks ) . the authors concluded that discontinuous drinking of a moderate amount of ethanol can be more harmful for the immune system than a continuous ethanol intake , as the model applied in the present study . those authors suggested that probably the discontinuous alcohol intake induces a greater stress as indicated by the searched immune indicators . therefore , future studies must be carried as to elucidate the isolate and combined effects of alcohol and stress over periodontal tissues . our data suggest that although stress and alcohol may be harmful separately , their association probably triggered different processes . the evaluation of these factors is relevant because the combination of stress and alcohol drinking is a frequent condition in human beings with periodontitis . although the association did not result as expected , the effects of alcohol drinking confirmed to be an important factor for the development of a periodontal disease . however , nonstressed animals who also consumed alcohol for the same period showed a pattern of periodontal breakdown even more severe . therefore , the factors of alcohol drinking and stress do not seem to show any synergic effect .
this study histometrically evaluated the effect of forced alcohol intake by stressed animals on the severity of ligature - induced periodontitis in rats . 
 thirty - two rats were randomly divided in four groups : group gal  alcohol and ligature ; group gasl  alcohol , chronic physical stress , and ligature ; gnc  negative control ; 
 gpc  positive control . 
 gal and gasl received 20% ethanol ad libitum , and gnc received water ad libitum for 60 days . 
 after 24 hours of exposition to alcohol intake  by gal and gasl 
 immobilization was applied as a chronic stressor in the gasl group for a two - month period , six times a week , in random hours . 
 the means of the respective groups were statistically compared ( analysis of variance and tukey tests , p < 0.05 ) . 
 the most severe periodontal breakdown was observed in nonstressed animals which drank alcohol ( gal ) , followed by stressed animals exposed to alcohol ( gasl ) . 
 gasl did not differ from the positive control group ( gpc ) . 
 the negative control group showed the lowest values of periodontal breakdown ( p < 0.05 ) . 
 conclusions . non - stressed alcohol consumer animals showed the most severe pattern of periodontal breakdown . 
 although stressed animals which were forced to drink alcohol showed poorer periodontal status than the negative controls , their results were similar to those of positive controls .